CN117385033A - Probe primer set for rapid typing identification of intraoperative glioma and detection kit - Google Patents
Probe primer set for rapid typing identification of intraoperative glioma and detection kit Download PDFInfo
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Abstract
The invention provides a probe primer group and a detection kit for rapid typing identification of intraoperative glioma. The upstream and downstream primers and probe sequences of the IDH1 probe primer group are respectively shown in SEQ ID NO. 1-3, the upstream and downstream primers and probe sequences of the TERT C228T probe primer group are respectively shown in SEQ ID NO. 4-6, the upstream and downstream primers and probe sequences of the TERT C250T probe primer group are respectively shown in SEQ ID NO. 7-9, and the LNA probe sequence in the C250T probe primer group is shown in SEQ ID NO. 10; the kit provided by the invention covers 1 mutation point (IDH 1R 132H) of the 132 th codon of IDH1 and 2 promoter mutation points (TERT C228T and TERT C250T) of the TERT gene, different specific primers are designed for each point, the specificity and the comprehensiveness of detection are improved, the possibility of missed detection is avoided, the extraction time of a brain glioma sample in operation is shortened to 8min, and the amplification time of a PCR reaction is shortened to 18min.
Description
Technical Field
The invention belongs to the technical field of medical biological detection, relates to a brain glioma typing identification marker, and particularly relates to a primer probe set for detecting promoter mutation of IDH1 genes and TERT genes and a detection kit containing the primer probe set.
Background
Diffuse gliomas are the most common tumors in the central nervous system, accounting for about 30% of intracranial tumors, accounting for more than 70% of tumors in neuroepithelial tissues. The tumor can be developed at any age, the incidence peaks are different from place to place, for example, astromas of the brain hemisphere often occur in adults of 30-50 years old, astromas of hypothalamus, cerebellum and brain bridge often occur in children and young, 80% of the astromas of the cerebellum occur frequently in adults, the average age is 24 years old, glioblastoma multiforme often occurs in adults of 45-55 years old.
Diffuse brain gliomas in adults are mainly divided into: astrocytoma, IDH mutant; oligodendrogliomas, isocitrate Dehydrogenase (IDH) mutations and 1p/19q co-deletions; glioblastoma, IDH wild type. Studies have shown that the greatest degree of safe excision can improve post-operative symptoms, quality of life, progression Free Survival (PFS) and total survival (OS) in glioma patients. At present, the main intervention technology in the operation aims at researching the tumor tissue boundary to seek to find out the tumor tissue infiltration boundary so as to realize the complete cutting of the tumor, such as navigation in the operation, magnetic resonance in the operation, fluorescence development in the operation, ultrasound in the operation and the like; on the other hand, the research on brain functional area identification is performed to avoid the damage to the brain functional area during operation, and the tumor is safely resected, such as nerve electrophysiological stimulation, and arousal during operation. The use of these techniques, alone or in combination, may enhance the PFS and OS in glioma patients.
In the traditional brain glioma operation treatment, a surgeon is difficult to completely remove diffuse brain glioma, and the removal range of glioma is always a research hot spot in the field of neurosurgery. In the precise medical era, personalized treatment of brain glioma patients with different molecular subtypes is a future trend. The 5 th edition of the tumor classification of the central nervous system of the world health organization provides a relatively clear diagnosis classification for different molecular subtypes (such as IDH mutation, 1p/19q co-deletion, TERT promoter mutation and the like), and gliomas with different diagnosis classifications correspond to different postoperative adjuvant treatment strategies and prognosis expectations, but the guidance of glioma operations is not clear.
Meanwhile, neurosurgeon needs to know the molecular pathology of the tumor in real time during operation, adjust the operation strategy in time, and balance between maximizing tumor resection and minimizing functional injury. In the traditional brain glioma operation treatment, neurosurgeons can report according to corresponding imaging and the like, meanwhile, detection such as frozen pathological sections and the like is carried out in operation through PCR, ARMS or immunohistochemical modes, and an individuation scheme is carried out on brain glioma patients with different molecular subtypes to carry out operation excision and treatment, but the detection modes are long in time consumption, relatively low in accuracy and sensitivity, and the balance between maximizing tumor excision and minimizing functional injury cannot be achieved. The advantages and disadvantages of the conventional detection technology are summarized in the following table 1:
TABLE 1 summary of advantages and disadvantages of conventional molecular typing detection methods
Disclosure of Invention
The invention relies on the research to provide a probe primer group and a detection kit for rapid extraction and parting identification of the brain glioma in the operation aiming at the defect of long detection time of the brain glioma in the operation at present, so that the detection time is shortened, the detection efficiency and stability are improved, and the detection sensitivity is improved.
In a first aspect of the present invention, a probe primer set for typing and identifying glioma is provided, and the method detects promoter mutations of human IDH1 gene and TERT gene, and has the following technical characteristics: the kit comprises an IDH1 probe primer group for detecting human IDH1R132H mutation sites, a TERT C228T probe primer group for detecting human TERT C228T and human TERT C250T mutation sites and a TERT C250T probe primer group.
The upstream and downstream primer sequences of the IDH1 probe primer group are shown as SEQ ID NO.1 and SEQ ID NO. 2, and the probe sequence is shown as SEQ ID NO. 3; the upstream and downstream primer sequences of the TERT C228T probe primer group are shown as SEQ ID NO.4 and 5, and the probe sequence is shown as SEQ ID NO.6;
the upstream and downstream primer sequences of the TERT C250T probe primer group are shown as SEQ ID NO.7 and 8, the probe sequence is shown as SEQ ID NO.9, and the LNA probe sequence in the C250T probe primer group is shown as SEQ ID NO.10.
Preferably, the probe primer group for typing and identifying the glioma also comprises an internal reference universal primer and a probe, wherein the sequences of the internal reference universal primer and the internal reference universal primer are shown as SEQ ID NO.11 and 12, and the sequences of the internal reference universal probe are shown as SEQ ID NO. 13.
Further preferred are IDH1 upstream primer and probe, TERT C228T upstream primer, TERT C250T upstream primer. LNA modification helps to increase the melting temperature of PCR primers or probes (T m Values) can significantly increase the ability to distinguish alleles, thereby improving hybridization efficiency and stability. Thus the sensitivity of the Taqman probe containing LNA bases is higher.
Specifically, the IDH1 upstream primer, the TERT C228T upstream primer and the last nucleotide from the 5' end in the sequence of the TERT C250T upstream primer are modified by LNA.
Further preferably, all probes contain a fluorescent reporter group at the 5 'end and a fluorescent quenching group at the 3' end, wherein the fluorescent groups attached to the internal reference universal probe are different from the fluorescent groups attached to the other probes. For example, FAM is selected as a fluorescent reporter gene at the 5 'end of the IDH1 probe, and BHQ is selected as a fluorescent quenching group at the 3' end; the fluorescent reporter gene at the 5 'end of the TERT C250T probe is FAM, and the fluorescent quenching group at the 3' end is MGB; the fluorescent reporter gene at the 5 'end of the TERT C228T probe is ROX, and the fluorescent quenching group at the 3' end is MGB; the fluorescent reporter gene at the 5 'end of the reference universal probe is VIC, and the fluorescent quenching group at the 3' end is BHQ2.
The invention provides a detection kit for rapid glioma typing identification in surgery, which has the following technical characteristics: comprises a probe primer group, a positive quality control product, a negative quality control product, a blank control liquid, a brain glioma tissue rapid extraction liquid and a PCR premix liquid.
Wherein the probe primer group is the probe primer group;
the positive quality control product is human genome DNA containing IDH1, TERT C228T or TERT C250T mutation rate of 1%, and is used for indicating whether a PCR reaction system is effective or not and whether a reaction program is correctly set or not;
the negative quality control is 10ng/μl of human genome DNA which does not contain IDH1, TERT C228T or TERT C250T mutation, and is used for indicating whether the PCR reaction system is effective and whether the reaction program is set correctly;
the brain glioma tissue rapid extraction solution comprises a NaOH solution with the concentration of 500mM and a Tris-HCl solution with the concentration of 100mM and the pH of 8.0; the brain glioma tissue rapid extract is developed and produced by Jiangsu Kuo biological medicine technology Co., ltd, and is tested in a laboratory to obtain the optimal brain glioma tissue rapid extract preferably.
The PCR premix comprises DNA polymerase, buffer, betaine, BSA, and Mg 2+ dNTPs. PCR premix can be purchased commercially and optimized and modified in the laboratory. The main enzyme raw material is Ace Taq DNA Polymerase (5U/. Mu.l) of Novain company, the product number is QN213-01 or QN213-02, and a certain amount of betaine, BSA and Mg are added into Buffer 2+, The optimal PCR premix is preferably obtained by mixing.
In a third aspect, the invention provides a method for rapid typing and identifying glioma during operation by using the detection kit, which comprises the following specific steps:
A. extraction of brain glioma tissue DNA
1 brain tissue cutting and tissue grinding
1) Cutting a proper amount of fresh brain tissue to be detected (3 mm multiplied by 3 mm) and placing the fresh brain tissue into a 1.5 ml centrifuge tube;
2) 50 microliters of 500mmol/L sodium hydroxide solution was added and the mixture was ground with a tissue grinding rod for 30 seconds, taking care that the grinding rod was not removed at the end of the grinding;
3) The polishing rod in the tube was rinsed while 300. Mu.l of 500mmol/L sodium hydroxide solution was added, and the used polishing rod was then removed;
4) Placing the centrifuge tube in a desktop palm centrifuge for transient instantaneous centrifugation;
5) 150 μl of mineral oil was added and covered on the surface of the ground mixture.
Note that: the related reagent consumables can be prepared in advance to shorten the operation time.
2 tissue lysis
1) Place 1.5 ml centrifuge tube on metal bath and perform the procedure: 98 ℃ for 5 minutes;
2) Taking out a 1.5 ml centrifuge tube from the metal bath, and placing the centrifuge tube on a tabletop palm centrifuge for transient instantaneous centrifugation;
3) 50 microliters of the middle aqueous phase was pipetted and diluted with 100 millimoles of Tris-HCl (pH 8.0) in a 1:20 ratio;
4) The dilutions can then be used in a rapid qPCR module.
B. Construction of PCR reaction System
The construction method of the IDH1 reaction system is as follows: mixing 10 ng/. Mu.l of PCR template and IDH1PCR reaction mixed solution according to a volume ratio of 1:9 to obtain an IDH1 reaction system;
the construction method of the C228T reaction system is as follows: mixing 10 ng/. Mu.l of PCR template and TERT C228T PCR reaction mixed solution according to a volume ratio of 1:9 to obtain a C228T reaction system;
the construction method of the C250T reaction system is as follows: mixing 10 ng/. Mu.l of PCR template and TERT C250T PCR reaction mixed solution in a volume ratio of 1:9 to obtain a C250T reaction system;
each detection has a positive quality control reaction system and a negative quality control reaction system, positive quality control products and negative quality control products are respectively mixed with PCR reaction mixed solution according to the volume ratio of 1:9 to obtain a positive quality control reaction system and a negative quality control reaction system,
in the specific practical operation, 20 mu L of total reaction system is adopted in each reaction system, 2 mu L of PCR template and 18 mu L of PCR reaction mixed solution are adopted in each reaction system.
The construction method of the PCR reaction mixed solution of IDH1, TERT C228T and TERT C250T comprises the following steps:
the construction method of the IDH1PCR reaction mixture is as follows: mixing the PCR premix, the IDH1 probe primer group, the internal reference universal primer and the probe to ensure that the final concentration of the primers is 0.2 mu mol/L respectively and the final concentration of the probe is 0.1 mu mol/L respectively;
the construction method of the TERT C228T PCR reaction mixture is as follows: mixing the PCR premix, the TERT C228T probe primer group, the internal reference universal primer and the probe to ensure that the final concentration of the primer at the upstream and downstream of the TERT C228T is 2.7 mu mol/L respectively and the final concentration of the TERT C228T probe is 0.25 mu mol/L; the final concentration of the upstream and downstream primers for internal reference is 0.2. Mu. Mol/L, respectively, the final concentration of the universal probe for internal reference is 0.1. Mu. Mol/L,
the construction method of the TERT C250T PCR reaction mixture is as follows: mixing the PCR premix, the TERT C250T probe primer group, the internal reference universal primer and the probe to ensure that the final concentration of the primer at the upstream and downstream of the TERT C250T is 1.5 mu mol/L respectively and the final concentration of the TERT C250T probe is 0.2 mu mol/L; the final concentration of the upstream and downstream primers for internal reference was 0.2. Mu. Mol/L, respectively, and the final concentration of the universal probe for internal reference was 0.1. Mu. Mol/L.
C. Rapid fluorescent quantitative PCR reaction
The fluorescence rapid PCR reaction procedure of the IDH1 and TERT reaction systems is as follows:
D. analysis of results
In a sample PCR reaction system, one primer probe composition and one inner reference primer probe composition in the IDH1 primer probe group, the TERT C228T primer probe group and the TERT C250T primer probe group are included in the same PCR system.
In a sample PCR reaction system, a fluorescent signal curve obtained by the internal reference primer probe composition is named as an internal reference amplification curve, an obtained Ct value is named as a Ct value of the internal reference amplification curve, and Ct values obtained by the IDH1 primer probe composition, the TERT C228T primer probe composition and the TERT C250T primer probe composition are named as Ct values of a sample amplification curve to be detected;
in an IDH1 positive quality control PCR reaction system, delta Ct Cationic property control product When the Ct value of the positive quality control FAM signal amplification curve-the Ct value of the internal reference VIC signal amplification curve is more than 8, the method indicates that the sample is realThe assay presents a problem and it is recommended to check if the reagents are overdue or if the PCR reaction procedure is correct. Delta Ct Cationic property control product Less than or equal to 8, which indicates that the experiment is effective. In the IDH1 negative quality control PCR reaction system, delta Ct Negative quality control product When the Ct value of the FAM signal amplification curve of the negative quality control product-the Ct value of the internal reference VIC signal amplification curve is less than or equal to 8, the experiment is problematic, and it is recommended to check whether the reagent is overdue or whether the PCR reaction procedure is correct. Delta Ct Negative quality control product > 8, indicating that the experiment was effective. Based on the effective experimental result, the internal reference VIC signal in the reaction system presents an S curve, and the internal reference Ct value is less than or equal to 32, so that the negative and positive of the IDH1 mutation of the sample to be detected can be judged: when the delta Ct in the IDH1 sample reaction system to be detected is less than or equal to 8, the sample reaction system to be detected is positive; when the delta Ct in the IDH1 sample reaction system to be detected is more than 8, the sample reaction system is negative, wherein delta Ct is equal to the Ct value of the FAM amplification curve of the sample to be detected-the Ct value of the internal reference VIC signal amplification curve;
in a PCR reaction system of a TERT C228T positive quality control product, ct Cationic property control product When Ct value of the positive quality control ROX signal amplification curve is > 35, it indicates that the experiment has a problem, and it is recommended to check whether the reagent is overdue or the PCR reaction procedure is correct. Ct is (Ct) Cationic property control product Ct value of the positive quality control ROX signal amplification curve is less than or equal to 35, which indicates that the experiment is effective. In the PCR reaction system of the TERT C228T negative quality control product, ct Negative quality control product When Ct value > 32 of reference VIC signal amplification curve in negative quality control, it indicates that the experiment is problematic, it is recommended to check whether the reagent is overdue or the PCR reaction procedure is correct. Ct is (Ct) Negative quality control product The Ct value of the reference VIC signal amplification curve of the negative quality control product is less than or equal to 32, which indicates that the experiment is effective. Based on the effective experimental result, the internal reference VIC signal in the reaction system presents an S curve, and the internal reference Ct value is less than or equal to 32, so that the negative and positive of the TERT C228T mutation of the sample to be detected can be judged: and when the ROX signal in the TERT C228T sample reaction system to be detected is Ct less than or equal to 35, the result is positive.
In a PCR reaction system of a TERT C250T positive quality control product, ct Cationic property control product When Ct value of positive quality control FAM signal amplification curve > 38, it indicates that the experiment is problematic, it is recommended to check whether the reagent is overdue or the PCR reaction procedure is correct. Ct is (Ct) Positive and negativeQuality control product Ct value of positive quality control FAM signal amplification curve is less than or equal to 38, which indicates that the experiment is effective. In the PCR reaction system of the TERT C250T negative quality control product, ct Negative quality control product When Ct value > 32 of reference VIC signal amplification curve in negative quality control, it indicates that the experiment is problematic, it is recommended to check whether the reagent is overdue or the PCR reaction procedure is correct. Ct is (Ct) Negative quality control product The Ct value of the reference VIC signal amplification curve of the negative quality control product is less than or equal to 32, which indicates that the experiment is effective. Based on the effective experimental result, the internal reference VIC signal in the reaction system presents an S curve, and the internal reference Ct value is less than or equal to 32, so that the negative and positive of the TERT C228T mutation of the sample to be detected can be judged: and when the FAM signal in the TERT C228T sample reaction system to be detected is Ct less than or equal to 38, the sample reaction system is positive.
The beneficial guarantee and effect of the invention are as follows:
the kit provided by the invention covers 1 mutation point of the 132 th codon of IDH1 (comprising IDH1R 132H) and 2 promoter mutation sites of TERT gene (comprising TERT C228T and TERT C250T), and different specific primers are designed for each site, so that the specificity and the comprehensiveness of detection are improved, and the possibility of missed detection is avoided.
According to the invention, a short probe design is selected in IDH1 and TERT promoter primer probe designs, a rapid extraction kit is selected in fresh brain glioma tissue extraction, and the sample extraction time is shortened to 8min; the rapid temperature raising and reducing module is selected in the amplification program, and the enzyme system is further optimized so as to adapt to the characteristic of rapid reaction, so that the whole experimental process is shortened, and the purpose of rapid detection is achieved. The amplification time of the existing kit in the market is mostly 1-2h, and the invention shortens the PCR amplification time to 18min.
LNA bases are respectively designed in the upstream primer sequences of IDH1 and TERT, and a common probe and an LNA modified probe are combined in a TERT C250T detection system, so that on one hand, the melting temperature (Tm value) of a PCR primer or a probe is increased, the capacity of distinguishing alleles can be obviously increased, the hybridization efficiency and stability are improved, and the sensitivity of the probe is also improved; on the other hand, the human TERT C250T adopts the common probe and the LNA probe at the same time, so that the hybridization characteristic and the influence on the melting point temperature are enhanced, and the probe containing the LNA can be designed more easily and flexibly to detect the problematic target sequence.
In the design process, an internal reference control system (ATCB gene) is selected, and an internal reference gene reaction solution (comprising an internal reference universal probe, an internal reference universal upstream primer and an internal reference universal downstream primer) is added into an IDH1 and TERT PCR reaction system, namely, a target gene PCR reaction and an internal reference gene PCR reaction run in the same PCR reaction system, so that the accuracy and the repeatability of an experiment are improved, and an indication effect is provided for judging an experimental result and judging whether the experimental operation is correct or not.
Drawings
FIG. 1 is a technical route of the present invention.
Fig. 2 and 3 show the IDH1 experimental group detection results: FIG. 2 shows FAM and VIC detection results of human genomic DNA without IDH1 gene mutation added in an amount of 20ng in a negative control group; FIG. 3 shows FAM and VIC detection results of a sample with an addition of 20ng, based on 1% mutation frequency of the standard DNA. Wherein FAM is the target gene, and VIC is the reference ATCB gene.
Fig. 4 to 6 show test results of TERT C228T test group: FIG. 4 shows the results of ROX and VIC detection of human genomic DNA without TERT C228T gene mutation added in an amount of 20ng in the negative control group; FIG. 5 shows the results of ROX and VIC detection of a sample with an addition of 20ng, based on the DNA standard with a mutation frequency of 1%; FIG. 6 shows the results of ROX and VIC detection of a sample with an addition of 20ng, based on the standard DNA with a mutation frequency of 10%. Wherein FAM is the target gene, and VIC is the reference ATCB gene.
Fig. 7 to 9 show test results of TERT C250T experimental group: FIG. 7 shows FAM and VIC detection results of human genomic DNA without TERT C250T gene mutation added in an amount of 20ng in a negative control group; FIG. 8 shows FAM and VIC detection results for a sample with a mutation frequency of 1% and an addition of 20 ng; FIG. 9 shows FAM and VIC detection results of a sample with a mutation frequency of 10% and an addition amount of 20 ng. Wherein FAM is the target gene, and VIC is the reference ATCB gene.
Detailed Description
The following examples of the present invention are provided to illustrate the detailed embodiments and specific operation procedures, but the scope of the present invention is not limited to the following examples.
The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
The nucleotide sequence of the IDH1 gene related to the invention is 209100953 th to 209119795 th positions of genbank accession number N C _000002, and month 9 of 2013.
The nucleotide sequence of the TERT gene was 1253167 to 1295068 of genbank accession number nc—000005, month 7 of 2008.
Example one, composition of kit and detection method
1. Detection kit
The detection kit for the intraoperative rapid glioma typing identification in the embodiment has the following technical characteristics: comprises a probe primer group, a positive quality control product, a negative quality control product, and also comprises a brain glioma tissue rapid extraction solution and a PCR premix solution.
The specific composition of each component is as follows:
(1) Probe primer set
The kit comprises an IDH1 probe primer group for detecting mutation sites of human IDH1R132H, a TERT C228T probe primer group and a TERT C250T probe primer group for detecting mutation sites of human TERT C228T and human TERT C250T, and an internal reference universal primer and a probe. The sequence composition of the primers and probes is shown in Table 2 below:
TABLE 2 sequence summarization of probe primer sets
Note that: fluorescent modification groups with italics of 5 'end and 3' end in the probe sequence are not part of the probe sequence, and the fluorescent groups connected with the internal reference universal probe are different from the fluorescent groups connected with other probes.
In the above sequences, the lower case is a normal base and the upper case is a base modified by LNA (locked nucleic acid). Specifically, the IDH1 upstream primer, the TERT C228T upstream primer and the last nucleotide from the 5' end in the sequence of the TERT C250T upstream primer are modified by LNA.
In the invention, the human TERT C250T adopts the common probe and the LNA probe at the same time, and the human IDH1R132H and the human TERT C228T adopt the common probe for the following reasons: the probe sequences of the human IDH1R132H and the human TERT C228T are relatively easy to design, the common probe can completely realize the specific detection, and the trouble that the detection reagent needs to be correspondingly adjusted is avoided; the human TERT C250T is not easy to detect by only adopting a common probe, the LNA probe strengthens the hybridization characteristic and the influence on the melting point temperature, and the probe containing the LNA can be designed to detect a problematic target sequence more easily and flexibly, and meanwhile, the thermal stability and the hybridization stability are improved.
(2) Cationic property control product
The positive quality control is human genome DNA with mutation rate of IDH1, TERT C228T or TERT C250T of 1%, and is used for indicating whether the PCR reaction system is effective and whether the reaction program is correctly set.
The specific preparation method of the IDH1, TERT C228T and TERT C250T positive quality control product comprises the following steps:
human genomic DNA containing IDH1 at the common mutation point IDH1 was mixed with wild-type (IDH 1R 132) human genomic DNA containing no IDH15 mutations (i.e., IDH1R132H, IDH1R 132C, IDH R132S, IDH R132G and IDH1R 132L) in proportions such that the IDH1 mutation frequency became 1%;
mixing human genome DNA containing TERT C228T mutation with wild type (TERT C228) human genome DNA without TERT C228T mutation in proportion to make the frequency of TERT C228T mutation become 1%;
human genomic DNA containing TERT C250T mutation was mixed with wild-type (TERT C250) human genomic DNA containing no TERT C250T mutation in proportions such that the frequency of TERT C250T mutation became 1%.
The total concentration of the positive quality control solution is 10 ng/. Mu.l, and the obtained solution is the positive quality control with mutation rates of IDH1, TERT C228T and TERT C250T of 1 percent respectively.
(3) Negative quality control product
The negative quality control is 10ng/μl of human genome DNA which does not contain IDH1, TERT C228T or TERT C250T mutation, and is used for indicating whether the PCR reaction system is effective and whether the reaction program is set correctly;
(4) The brain glioma tissue rapid extraction solution comprises a NaOH solution with the concentration of 500mM and a Tris-HCl solution with the concentration of 100mM and the pH of 8.0; the brain glioma tissue rapid extract is developed and produced by Jiangsu Kuo biological medicine technology Co., ltd, and is tested in a laboratory to obtain the optimal brain glioma tissue rapid extract preferably.
(5) The PCR premix comprises DNA polymerase, buffer, betaine, BSA, and Mg 2+ dNTPs. The PCR premix can be purchased commercially, optimized and modified in laboratory, and the main enzyme raw material is Ace Taq DNA Polymerase (5U/. Mu.l) of Novain company, with the product number of QN213-01 or QN213-02, and Buffer is prepared by adding betaine, BSA, and Mg in certain amounts 2+ The optimal PCR premix is preferably obtained by mixing.
2. Detection method
The technical route of the invention is shown in fig. 1:
A. extraction of brain glioma tissue DNA
1. Brain tissue cutting and tissue milling (operation time in 2 minutes based on single sample)
1) Cutting a proper amount of fresh brain tissue to be detected (3 mm multiplied by 3 mm) and placing the fresh brain tissue into a 1.5 ml centrifuge tube;
2) 50. Mu.L of 500mmol/L sodium hydroxide solution was added and the mixture was ground with a tissue grinding rod for 30 seconds, taking care that the grinding rod was not taken out at the end of the grinding;
3) The polishing rod in the tube was rinsed while 300. Mu.L of 500mmol/L sodium hydroxide solution was added thereto, and then the used polishing rod was removed;
4) Placing the centrifuge tube in a desktop palm centrifuge for transient instantaneous centrifugation;
5) 150 μl of mineral oil was added and covered on the surface of the ground mixture.
Note that: the related reagent consumables can be prepared in advance to shorten the operation time.
2. Tissue lysis (based on a single sample, the operating time is within 6 minutes)
1) Place 1.5 ml centrifuge tube on metal bath and perform the procedure: 98 ℃ for 5 minutes;
2) Taking out a 1.5 ml centrifuge tube from the metal bath, and placing the centrifuge tube on a tabletop palm centrifuge for transient instantaneous centrifugation;
3) 50 μl of the intermediate aqueous phase was pipetted and 100mM Tris-HCl (pH 8.0) was used at a ratio of 1: 20;
4) The dilutions can then be used in a rapid qPCR module.
Note that: the metal bath should be heated to the desired temperature in advance. The related reagent consumables can be prepared in advance to shorten the operation time.
B. Construction of PCR reaction System
1) Construction of PCR reaction mixture
The IDH1PCR reaction mixture was prepared by adding 10. Mu.M of the following substances to the PCR premix, see Table 3 below:
TABLE 3 composition of IDH1PCR reaction mixture
The TERT C228T PCR reaction mixture was prepared by adding the following substances to the PCR premix at a concentration of 100 μm TERT C228T primer and a remaining concentration of 10 μm, see table 4 for details:
table 4 substance of the mixture of TERT C228T PCR reactions
| Name of the name | Volume (ul) | Final concentration (uM) |
| TERTC228T upstream specific primer | 0.54 | 2.7uM |
| TERTC228T downstream specific primer | 0.54 | 2.7uM |
| TERTC228T probe | 0.5 | 0.25uM |
| Upstream primer for internal reference | 0.4 | 0.2uM |
| Universal downstream primer for internal reference | 0.4 | 0.2uM |
| Internal reference universal probe | 0.2 | 0.1uM |
The TERT C250T PCR reaction mixture was prepared by adding the following substances to the PCR premix at a concentration of 100. Mu.M for the TERT C250T primer and 10. Mu.M for the rest, see Table 5 for details:
table 5 substance of the mixture of TERT and C250T PCR reactions
| Name of the name | Volume (ul) | Final concentration (uM) |
| TERTC250T upstream specific primer | 0.3 | 1.5μM |
| TERTC250T downstream specific primer | 0.3 | 1.5μM |
| TERTC250T-LNA | 0.4 | 0.2μM |
| TERTC250T probe | 0.4 | 0.2μM |
| Upstream primer for internal reference | 0.4 | 0.2uM |
| Universal downstream primer for internal reference | 0.4 | 0.2uM |
| Internal reference universal probe | 0.2 | 0.1uM |
2) Construction of PCR reaction System
The construction method of the IDH1 reaction system is as follows: mixing 2 mu L of 10 ng/. Mu.l of PCR template and 18 mu. L, IDH1 of PCR reaction mixture to obtain an IDH1 reaction system;
the construction method of the C228T reaction system is as follows: mixing 2 mu L of 10 ng/mu L of PCR template with 18 mu L of TERT C228T PCR reaction mixed solution to obtain a C228T reaction system;
the construction method of the C250T reaction system is as follows: mixing 2 mu L of 10 ng/mu L of PCR template with 18 mu L of TERT C250T PCR reaction mixed solution to obtain a C250T reaction system;
each detection has a positive quality control reaction system and a negative quality control reaction system, and 2 mu L of positive quality control product and 18 mu L of negative quality control product are respectively mixed with the PCR reaction mixed solution to obtain a positive quality control reaction system and a negative quality control reaction system, and the specific reference is shown in Table 6:
TABLE 6 composition of cationic and anionic controlled reaction systems
| Reaction system | Positive quality control reaction system | Negative quality control reaction system |
| 18 μl of PCR reaction mixture | 18 μl of PCR reaction mixture | 18 μl of PCR reaction mixture |
| Sample PCR template at 10 ng/. Mu.l 2. Mu.l | Cationic quality control 2. Mu.l | Negative quality control 2. Mu.l |
C. Rapid fluorescent quantitative PCR reaction
Selection of real-time fluorescent quantitative PCR instrument (model Nuo Zhan Turbo 16): IDH1 selects FAM and VIC channels, TERT C228T selects ROX and VIC channels, TERT C250T selects FAM and VIC channels.
The PCR conditions were as follows:
TABLE 7 fluorescent Rapid PCR reaction procedure for IDH1, TERT reaction systems
D. Analysis of results
The Ct value in the invention refers to the cycle number when the fluorescent signal in the reaction system reaches the set baseline value, the baseline value of the amplification curve is set to 10000, the cycle number is 3-12, and the Ct value is determined under the condition; in IDH1 and TERT C250T, the VIC signal detects the internal reference amplification condition, the FAM signal detects blank control, positive quality control and the amplification condition of the PCR template of the sample to be detected, the VIC signal in TERT C250T detects the internal reference amplification condition, and the FAM signal detects positive quality control and the amplification condition of the PCR template of the sample to be detected.
The IDH1 reaction system, the C228T reaction system and the C250T reaction system are set to be consistent in amplification curve, cycle number condition, delta Ct judgment value, negative quality control product reaction system and positive quality control product reaction system.
In a sample PCR reaction system, a fluorescent signal curve obtained by the internal reference primer probe composition is named as an internal reference amplification curve, an obtained Ct value is named as a Ct value of the internal reference amplification curve, and Ct values obtained by the IDH1 primer probe composition, the TERT C228T primer probe composition and the TERT C250T primer probe composition are named as Ct values of a sample amplification curve to be detected;
in an IDH1 positive quality control PCR reaction system, delta Ct Cationic property control product When the Ct value of the positive quality control FAM signal amplification curve-the Ct value of the internal reference VIC signal amplification curve > 8, the experiment is problematic, and it is recommended to check whether the reagent is overdue or the PCR reaction procedure is correct. Delta Ct Cationic property control product Less than or equal to 8, which indicates that the experiment is effective. In the IDH1 negative quality control PCR reaction system, delta Ct Negative quality control product When the Ct value of the FAM signal amplification curve of the negative quality control product-the Ct value of the internal reference VIC signal amplification curve is less than or equal to 8, the experiment is problematic, and it is recommended to check whether the reagent is overdue or whether the PCR reaction procedure is correct. Delta Ct Negative quality control product > 8, indicating that the experiment was effective. Based on the effective experimental result, the internal reference VIC signal in the reaction system presents an S curve, and the internal reference Ct value is less than or equal to 32, so that the negative and positive of the IDH1 mutation of the sample to be detected can be judged: when the delta Ct in the IDH1 sample reaction system to be detected is less than or equal to 8, the sample reaction system to be detected is positive; when the delta Ct in the IDH1 sample reaction system to be detected is more than 8, the sample reaction system is negative, wherein delta Ct is equal to the Ct value of the FAM amplification curve of the sample to be detected-the Ct value of the internal reference VIC signal amplification curve;
in a PCR reaction system of a TERT C228T positive quality control product, ct Cationic property control product When Ct value of the positive quality control ROX signal amplification curve is > 35, it indicates that the experiment has a problem, and it is recommended to check whether the reagent is overdue or the PCR reaction procedure is correct. Ct is (Ct) Cationic property control product Ct value of the positive quality control ROX signal amplification curve is less than or equal to 35, which indicates that the experiment is effective. In the PCR reaction system of the TERT C228T negative quality control product, ct Negative quality control product When Ct value > 32 of reference VIC signal amplification curve in negative quality control, it indicates that the experiment is problematic, it is recommended to check whether the reagent is overdue or the PCR reaction procedure is correct. Ct is (Ct) Negative quality control product The Ct value of the reference VIC signal amplification curve of the negative quality control product is less than or equal to 32, which indicates that the experiment is effective. Based on the effective experimental result, the internal reference VIC signal in the reaction system presents an S curve, and the internal reference Ct value is less than or equal to 32, so that the negative and positive of the TERT C228T mutation of the sample to be detected can be judged: when the ROX signal in the TERT C228T sample reaction system to be detected is Ct less than or equal to 35, the ROX signal isPositive.
In a PCR reaction system of a TERT C250T positive quality control product, ct Cationic property control product When Ct value of positive quality control FAM signal amplification curve > 38, it indicates that the experiment is problematic, it is recommended to check whether the reagent is overdue or the PCR reaction procedure is correct. Ct is (Ct) Cationic property control product Ct value of positive quality control FAM signal amplification curve is less than or equal to 38, which indicates that the experiment is effective. In the PCR reaction system of the TERT C250T negative quality control product, ct Negative quality control product When Ct value > 32 of reference VIC signal amplification curve in negative quality control, it indicates that the experiment is problematic, it is recommended to check whether the reagent is overdue or the PCR reaction procedure is correct. Ct is (Ct) Negative quality control product The Ct value of the reference VIC signal amplification curve of the negative quality control product is less than or equal to 32, which indicates that the experiment is effective. Based on the effective experimental result, the internal reference VIC signal in the reaction system presents an S curve, and the internal reference Ct value is less than or equal to 32, so that the negative and positive of the TERT C228T mutation of the sample to be detected can be judged: and when the FAM signal in the TERT C228T sample reaction system to be detected is Ct less than or equal to 38, the sample reaction system is positive.
Example 2 1% Gene mutation Standard DNA Rapid qPCR reaction
1. Preparation of 1% mutation standard sample:
IDH1, TERT C228T, TERT C250T PCR reaction system: 100% mutant standard DNA of IDH1, TERT C228T and TERT C250T was taken out respectively, the concentration after quantification was 10 ng/. Mu.l, and 10 ng/. Mu.l of negative quality control was used for dilution to 1% mutant standard DNA.
2. Construction of PCR reaction System
Mu.l of IDH11% mutant standard DNA (10 ng/. Mu.l) and negative controls were added to 18. Mu.l of IDH1 reaction mixture, respectively, in a total volume of 20. Mu.l.
Mu.l of TERT C228T 1% mutant standard DNA (10 ng/. Mu.l) and negative quality control were added to 18. Mu.l of the IDH1 reaction mixture, respectively, in a total volume of 20. Mu.l.
Mu.l of TERT C250T 1% mutant standard DNA (10 ng/. Mu.l) and negative quality control were added to 18. Mu.l of the IDH1 reaction mixture, respectively, in a total volume of 20. Mu.l.
3. PCR amplification
The PCR reaction tube was placed in a rapid fluorescent quantitative PCR instrument and the positional information was recorded and instrument related parameters were set according to the qPCR reaction procedure in tables 7 and 8. The detection results are shown in fig. 2-9, and the results show that the detection kit provided by the invention can rapidly and accurately detect standard products with mutation rates of IDH1, TERT C228T and TERT C250T of 1%.
EXAMPLE 3 methodological validation of the kit of the invention to detect comparative NGS
The kit prepared in example 1 was used to perform rapid fluorescent PCR detection (rapid qPCR detection) in accordance with the detection method in example 1, while high throughput sequencing (NGS) methodology validation was performed. The data is validated against NGS methodology as follows: and randomly selecting frozen tissue samples of 10 brain glioma patients, and carrying out a comparison experiment of the kit provided by the invention and an NGS methodology in parallel to obtain a verification result of the compliance rate of the kit detection and the NGS methodology. The results of the verification are shown in table 8 below:
TABLE 8 comparison of test results of the kit and NGS methodology provided by the present invention
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Remarks: WT: wild type, MUT: mutant type.
In the IDH1PCR reaction system and NGS sequencing, the wild type is IDH1R132, and the mutant type is IDH1;
in a TERT C228T PCR reaction system and NGS sequencing, the wild type is TERT C228, and the mutant type is TERT C228T;
in the TERT C250T PCR reaction system and NGS sequencing, the wild type is TERT C250, and in the TERT C228TPCR reaction system, the mutant type is TERT C250T.
From the clinical results of frozen tissue samples of 10 brain glioma patients obtained at present, 10 effective detection data are obtained, and the results prove that the wild type, mutant type and overall coincidence rate of IDH1, TERT C228T and TERT C250T are all 100%.
In summary, the kit provided by the invention adopts a rapid fluorescence quantitative PCR method to detect 1 mutation site related to the 132 th codon of the existing IDH1 gene and 2 promoter mutation sites of the TERT gene, and different specific primers are designed aiming at each site, so that the specificity and the comprehensiveness of detection are improved, and the possibility of missed detection is avoided.
In terms of detection time, the rapid fluorescence quantitative PCR method adopted by the invention combines the application technology in the molecular diagnosis field with an amplification inhibition mutation system (amplification refractory mutation system, ARMS), so that the aim of distinguishing and detecting alleles is fulfilled, meanwhile, the sensitivity is improved, the whole experimental process is shortened, and the PCR amplification program time is shortened to 18min.
In addition, in the traditional operation process, doctors carry out confirmation of a brain glioma excision scheme and operation excision treatment based on reports such as imaging and detection of frozen pathological sections, the detection mode is long in time consumption, accuracy and sensitivity are relatively low, and balance between maximizing tumor excision and minimizing functional injury cannot be achieved. The invention reduces the detection time to 26min, and the sensitivity and specificity of the detection are far higher than those of other detection modes. A balance is struck between maximizing tumor resection and minimizing functional impairment.
While the embodiments of the present invention have been described in detail, the present invention is not limited to the embodiments, and various equivalent modifications and substitutions can be made by those skilled in the art without departing from the spirit of the present invention, and these equivalent modifications and substitutions are intended to be included in the scope of the present invention as defined in the appended claims.
Claims (10)
1. A probe primer set for rapid typing identification of brain glioma detects promoter mutation of human IDH1 gene and TERT gene, which is characterized by comprising an IDH1 probe primer set for detecting human IDH1R132H mutation site, a TERT C228T probe primer set for detecting human TERT C228T and human TERT C250T mutation site and a TERT C250T probe primer set,
the upstream and downstream primer sequences of the IDH1 probe primer group are shown as SEQ ID NO.1 and SEQ ID NO. 2, and the probe sequence is shown as SEQ ID NO. 3;
the upstream and downstream primer sequences of the TERT C228T probe primer group are shown as SEQ ID NO.4 and 5, and the probe sequence is shown as SEQ ID NO.6;
the upstream and downstream primer sequences of the TERT C250T probe primer group are shown as SEQ ID NO.7 and 8, the probe sequence is shown as SEQ ID NO.9, and the LNA probe sequence is shown as SEQ ID NO.10.
2. The probe primer set for rapid typing identification of brain glioma according to claim 1, further comprising an internal reference primer and a probe,
the sequences of the upper and lower primers of the internal reference general probe are shown as SEQ ID NO.11 and 12, and the sequence of the internal reference general probe is shown as SEQ ID NO. 13.
3. The probe primer set for rapid typing identification of brain glioma according to claim 2, wherein:
wherein the IDH1 upstream primer, the TERT C228T upstream primer and the TERT C250T upstream primer sequences are all modified by LNA,
the 5 'end of all the probes contains a fluorescence report group, the 3' end contains a fluorescence quenching group, and the fluorescence groups connected with the internal reference universal probes are different from the fluorescence groups connected with other probes.
4. The probe primer set for rapid typing identification of brain glioma according to claim 3, wherein:
the IDH1 upstream primer, the TERT C228T upstream primer and the last nucleotide from the 5' end in the sequence of the TERT C250T upstream primer are subjected to LNA modification.
5. A detection kit for rapid typing and identification of intraoperative glioma is characterized by at least comprising a probe primer group, a positive quality control product and a negative quality control product,
wherein the probe primer set is the probe primer set according to any one of claims 1 to 4.
6. The detection kit for rapid typing identification of intraoperative glioma according to claim 5, further comprising a rapid glioma tissue extraction solution and a PCR premix solution,
the brain glioma tissue rapid extraction solution comprises a NaOH solution with the concentration of 500mM, a Tris-HCl solution with the concentration of 100mM and the pH of 8.0;
the PCR premix comprises DNA polymerase, buffer, betaine, BSA and Mg 2+ 、dNTPs。
7. The detection kit for rapid typing identification of intraoperative glioma according to claim 5, wherein:
wherein the negative quality control is human genome DNA which does not contain IDH1, TERT C228T or TERT C250T mutation; the positive quality control product is human genome DNA containing IDH1, TERT C228T or TERT C250T mutation rate of 1%.
8. The detection kit for rapid typing identification of intraoperative glioma according to claim 6, wherein:
wherein, the steps of tissue DNA extraction by adopting the brain glioma tissue rapid extraction solution are as follows:
A. brain tissue cutting and tissue grinding
1) A proper amount of fresh brain tissue to be measured is cut and put into a centrifuge tube with the volume of 1.5 milliliters,
2) Adding 50. Mu.L of 500mmol/L sodium hydroxide solution, grinding for 30 seconds by using a tissue grinding rod, taking out the grinding rod at the end of grinding,
3) 300. Mu.l of 500mmol/L sodium hydroxide solution were added and the polishing rod in the tube was rinsed, and the used polishing rod was then removed,
4) Placing the centrifuge tube on a desktop palm centrifuge for transient instantaneous centrifugation, adding 150 microliters of mineral oil, and covering the surface of the grinding mixture;
B. tissue lysis
1) Place 1.5 ml centrifuge tube on metal bath and perform the procedure: 98 ℃ for 5 minutes;
2) Taking off the centrifuge tube from the metal bath, and placing the centrifuge tube on a tabletop palm centrifuge for transient instantaneous centrifugation;
3) 50. Mu.L of the middle aqueous phase was pipetted and diluted with Tris-HCl pH 8.0 in a ratio of 1:20, the dilutions subsequently being used in a rapid qPCR module,
when the PCR reaction is carried out, the construction method of the IDH1 reaction system is as follows: mixing 10 ng/. Mu.l of PCR template and IDH1PCR reaction mixed solution according to a volume ratio of 1:9 to obtain an IDH1 reaction system; the construction method of the C228T reaction system is as follows: mixing 10 ng/. Mu.l of PCR template and TERT C228T PCR reaction mixed solution according to a volume ratio of 1:9 to obtain a C228T reaction system; the construction method of the C250T reaction system is as follows: mixing 10 ng/. Mu.l of PCR template and TERT C250T PCR reaction mixed solution in a volume ratio of 1:9 to obtain a C250T reaction system;
each detection has a positive quality control reaction system and a negative quality control reaction system, and positive quality control products and negative quality control products are respectively mixed with the PCR reaction mixed solution according to the volume ratio of 1:9, so that a positive quality control reaction system and a negative quality control reaction system are obtained.
9. The detection kit for rapid typing identification of intraoperative glioma according to claim 8, wherein:
the construction method of the IDH1PCR reaction mixed solution comprises the following steps: mixing the PCR premix, the IDH1 probe primer group, the internal reference universal primer and the probe to ensure that the final concentration of the primers is 0.2 mu mol/L respectively and the final concentration of the probe is 0.1 mu mol/L respectively;
the construction method of the TERT C228T PCR reaction mixture is as follows: mixing the PCR premix, the TERT C228T probe primer group, the internal reference universal primer and the probe to ensure that the final concentration of the primer at the upstream and downstream of the TERT C228T is 2.7 mu mol/L respectively and the final concentration of the TERT C228T probe is 0.25 mu mol/L; the final concentration of the upstream and downstream primers for internal reference is 1.5. Mu. Mol/L, respectively, the final concentration of the universal probe for internal reference is 0.3. Mu. Mol/L,
the construction method of the TERT C250T PCR reaction mixture is as follows: mixing the PCR premix, the TERT C250T probe primer group, the internal reference universal primer and the probe to ensure that the final concentration of the primer at the upstream and downstream of the TERT C250T is 1.5 mu mol/L respectively and the final concentration of the TERT C250T probe is 0.2 mu mol/L; the final concentration of the TERT C250T LNA probe is 0.2 mu mol/L; the final concentration of the upstream and downstream primers for internal reference was 1.5. Mu. Mol/L, respectively, and the final concentration of the universal probe for internal reference was 0.3. Mu. Mol/L.
10. The test kit for intraoperative rapid glioma typing identification of claim 8, wherein:
the fluorescence rapid PCR reaction program of the IDH1 and TERT reaction systems is as follows:
in the IDH1 reaction system, after the reaction is completed, the reaction is carried out according to delta Ct Cationic property control product Ct value of positive quality control amplification curve-Ct value of internal reference amplification curve to calculate Δct Cationic property control product According to DeltaCt Negative quality control product Ct value of amplification curve of negative quality control material-Ct value of internal reference amplification curve, delta Ct is calculated Negative quality control product ，
When delta Ct Cationic property control product ≤8，ΔCt Negative quality control product At > 8, the experiment was effective; under the effective experimental condition, the internal reference fluorescent signal in the reaction system presents an S-shaped curve, and when the internal reference Ct value is less than or equal to 32, the negative and positive of the IDH1 mutation of the sample to be detected are judged, and the detection is carried out according to delta Ct IDH1 Ct value of amplification curve of IDH1 sample system-Ct value of internal reference amplification curve to calculate Δct IDH1 ，ΔCt IDH1 A threshold of 8;
in a TERT C228T reaction system, ct Cationic property control product Ct value, ct of the positive quality control ROX signal amplification curve Negative quality control product Ct value of reference VIC signal amplification curve for negative quality control, when Ct Cationic property control product ≤35，Ct Negative quality control product The experiment is effective when the temperature is less than or equal to 32; in the experimentUnder the effective condition, an internal reference fluorescent signal in the reaction system presents an S curve, and when the internal reference Ct value is less than or equal to 32, the negative and positive of the TERT C228T mutation of the sample to be detected are judged based on the Ct value of the ROX signal in the reaction system of the sample to be detected of TERT C228T, and the threshold value of the Ct value is 35;
in a TERT C250T reaction system, ct Cationic property control product Ct value, ct of positive quality control FAM signal amplification curve Negative quality control product Ct value of reference VIC signal amplification curve for negative quality control, when Ct Cationic property control product ≤38，Ct Negative quality control product The experiment is effective when the temperature is less than or equal to 32; under the experimental effective condition, an internal reference fluorescent signal in a reaction system presents an S curve, and when the internal reference Ct value is less than or equal to 32, the negative and positive of the mutation of TERT C250T of a sample to be detected are judged based on the Ct value of the FAM signal in the reaction system of the sample to be detected of TERT C228T, and the threshold value of the Ct value is 38.
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