CN113337587A - Rapid sensitive detection method for NOTCH2NLC gene GGC repetitive sequence and application thereof - Google Patents

Rapid sensitive detection method for NOTCH2NLC gene GGC repetitive sequence and application thereof Download PDF

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CN113337587A
CN113337587A CN202110850961.1A CN202110850961A CN113337587A CN 113337587 A CN113337587 A CN 113337587A CN 202110850961 A CN202110850961 A CN 202110850961A CN 113337587 A CN113337587 A CN 113337587A
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CN113337587B (en
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孙淼
王彬
徐兴顺
鲁礼魁
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First Affiliated Hospital of Suzhou University
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Abstract

The invention relates to the field of medicine, in particular to a rapid sensitive detection method of a NOTCH2NLC gene GGC repetitive sequence and application thereof. Firstly, a primer composition for detecting a GGC repetitive sequence of a NOTCH2NLC gene is disclosed, which comprises the following components: the nucleotide sequence is shown as SEQ ID NO: 1, and the nucleotide sequence of the upstream primer is shown as SEQ ID NO: 2, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 3, or a third primer. The invention discloses a novel primer composition for detecting a sample with high GGC repetition number, optimizes a PCR detection program, is more sensitive to the detection of the sample with high GGC repetition number, can accurately quantify and complete the detection more quickly, shortens the PCR time from 10 hours to within 2 hours, effectively reduces the workload of operators and reduces the detection cost.

Description

Rapid sensitive detection method for NOTCH2NLC gene GGC repetitive sequence and application thereof
Technical Field
The invention relates to the field of medicine, in particular to a rapid sensitive detection method of a NOTCH2NLC gene GGC repetitive sequence and application thereof.
Background
Neuron endonuclear inclusion disease (NIID) is a very rare disease, early case reports are common in European and American people, and children and teenagers are common, and later Asian adults are found to be more common. The clinical manifestations of NIID are complex and diverse and include dementia, ataxia, behavioral abnormalities, tremors, epilepsy, and the like. The abnormal repeated amplification of 5' UTR GGC in NOTCH2NLC gene is related to NIID in 2019, and then the abnormal repeated amplification is related to various neurodegenerative diseases such as Alzheimer disease, frontotemporal dementia and the like[1]。
Currently, the diagnosis of NIID mainly involves classical P62 and UB staining positive eosinophilic endonuclears and NOTCH2NLC gene detection, and diagnosis is not difficult. Based on PCR amplification of NOTCH2NLC gene, the resolution ratio of the capillary electrophoresis detection amplification fragment is higher than that of the traditional agarose gel electrophoresis, but because the CG proportion of the amplification fragment is too high and is difficult to increase along with the increase of the number of times of repetition, most of PCR primers are usually designed, because the PCR reaction system is not adjusted to the optimal condition, the detection result can be misjudged, and meanwhile, the PCR time is over 10 hours[2]. Therefore, the invention of a novel rapid sensitive detection method of the NOTCH2NLC gene GGC repetitive sequence is needed.
Disclosure of Invention
The invention aims to disclose a novel detection method, which is also called triple Repeat Primary PCR (TP-PCR) by a three-primer PCR method. The TP-PCR method is different from the traditional PCR amplification method, and 3 primers are added in the reaction. The TP-PCR method of the NOTCH2NLC gene adds a GGC sequence primer on the basis of general PCR amplification, is more sensitive to the detection of a sample with high GGC repetition number, can accurately quantify and complete the detection more quickly, shortens the PCR time from 10 hours to within 2 hours (1 hour and 40 minutes), effectively reduces the workload of operators and reduces the detection cost.
Specifically, the technical scheme of the invention is as follows:
the invention discloses a primer composition for detecting a GGC repetitive sequence of a NOTCH2NLC gene, which comprises the following components: the nucleotide sequence is shown as SEQ ID NO: 1, and the nucleotide sequence of the upstream primer is shown as SEQ ID NO: 2, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 3, or a third primer.
In a second aspect, the invention discloses an amplification reaction system for amplifying a GGC repeat sequence of a NOTCH2NLC gene, wherein the amplification reaction system comprises the primer composition.
The third aspect of the invention discloses a kit for rapidly detecting a GGC repetitive sequence of a NOTCH2NLC gene, which comprises the primer composition or the amplification reaction system.
The fourth aspect of the invention discloses a method for rapidly detecting a NOTCH2NLC gene GGC repetitive sequence by using the amplification reaction system or the kit, wherein the PCR reaction program for amplifying the NOTCH2NLC gene GGC repetitive sequence is as follows:
reacting for 10min at the temperature of 98 ℃; followed by reaction at 98 ℃ for 30sec, at 66 ℃ for 1min (0.5 ℃ per cycle), at 68 ℃ for 1min, for a total of 20 cycles; then reacting at 98 ℃ for 30sec, at 56 ℃ for 30sec, and at 68 ℃ for 1min, and totaling 15 cycles; finally 68 ℃ for 10 minutes. The adjustment for all cycling steps was 0.5 ℃/sec.
Preferably, the method comprises:
obtaining genome DNA of a sample to be detected;
amplifying the NOTCH2NLC gene by using the amplification reaction system or the kit through PCR;
and detecting the repeated sequence GGC of the NOTCH2NLC gene in the obtained amplification product.
Preferably, the repeated sequence GGC of the NOTCH2NLC gene in the amplification product is detected by capillary electrophoresis.
The fifth aspect of the invention discloses the application of the primer composition, the amplification reaction system, the kit or the method in the medical field.
Preferably, the application is the application of the primer composition, the amplification reaction system, the kit or the method in auxiliary prediction and/or diagnosis of the neuron endonucleosome diseases.
On the basis of the common general knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily without departing from the concept and the protection scope of the invention.
Compared with the prior art, the invention has the following remarkable advantages and effects:
the invention discloses a novel primer composition for detecting a sample with high GGC repetition number, optimizes a PCR detection program, is more sensitive to the detection of the sample with high GGC repetition number, can accurately quantify and complete the detection more quickly, shortens the PCR time from 10 hours to within 2 hours, effectively reduces the workload of operators and reduces the detection cost.
Drawings
FIG. 1 is a schematic diagram of the design of a NOTCH2NLC gene 5' UTR GGC primer;
FIG. 2 is a schematic diagram showing the comparison of capillary electrophoresis effects of DNA samples of positive patients detected by different primers;
FIG. 3 is a diagram showing the comparison of capillary electrophoresis effect sensitivity of DNA samples of positive patients detected by different primers;
FIG. 4 is a schematic diagram showing the comparison of capillary electrophoresis effects under different PCR detection procedures;
FIG. 5 is a schematic diagram showing the electrophoresis effect of capillary vessels in 10 positive patients;
FIG. 6 is a diagram showing the effect of capillary electrophoresis in 10 negative cases.
Detailed Description
The technical solutions of the present invention are described in detail below with reference to the drawings and the embodiments, but the present invention is not limited to the scope of the embodiments.
The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions. The reagents and starting materials used in the present invention are commercially available.
Example 1
The embodiment discloses a method for rapidly detecting a GGC repetitive sequence of a NOTCH2NLC gene, which specifically comprises the following steps:
firstly, obtaining genome DNA of a sample to be detected;
secondly, amplifying a NOTCH2NLC gene by using an amplification reaction system;
and thirdly, detecting a NOTCH2NLC gene repetitive sequence GGC in the obtained amplification product.
Wherein the amplification reaction system comprises a primer composition, the primer composition consists of an upstream primer, a downstream primer and a third primer of a non-human gene sequence, and is shown in figure 1, and the specific sequence is shown in table 1.
TABLE 1
Figure 159847DEST_PATH_IMAGE001
The amplification reaction system is shown in Table 2 below.
TABLE 2
Figure DEST_PATH_IMAGE003
The cited document 2 procedure, i.e. the amplification procedure, is:
reacting for 10min at the temperature of 98 ℃; followed by a reaction at 98 ℃ for 30sec, at 66 ℃ for 1min (0.5 ℃ per cycle), and at 68 ℃ for 8min for a total of 16 cycles; then reacting at 98 ℃ for 30sec, at 58 ℃ for 1min and at 68 ℃ for 8min, and totaling 29 cycles; the final 68 ℃ was continued for 10 minutes, with all cycle steps adjusted to 0.5 ℃/sec.
The results of experiments using the primers in the literature as comparative examples and the primers in Table 1 as tests are shown in FIG. 2, and it can be seen from FIG. 2,
example 2
The method of this example is different from example 1 only in that the amplification procedure is as follows:
reacting for 10min at the temperature of 98 ℃; followed by reaction at 98 ℃ for 30sec, at 66 ℃ for 1min (0.5 ℃ per cycle), at 68 ℃ for 1min, for a total of 20 cycles; then reacting at 98 ℃ for 30sec, at 56 ℃ for 30sec, and at 68 ℃ for 1min, and totaling 15 cycles; finally 68 ℃ for 10 minutes. The adjustment of all the circulation steps is 0.5 ℃/s, and the new program is obtained.
The discovery that the novel program of the inventive primer (shown in table 1 above) well solves the PCR amplification of the 5'UTR GGC abnormal repeat in NOTCH2NLC gene, and as shown in FIG. 4 below, the discovery that the program of 1 hour and 40 minutes can complete the PCR amplification of the 5' UTR GGC abnormal repeat in NOTCH2NLC gene.
Example 3
This example investigated the sensitivity of the inventive primers to aberrant repeat amplification of 5' UTR GGC in different template positive control NOTCH2NLC genes, with the results shown in FIG. 3. After the DNA sample (5 ng-300 ng) of a positive control patient is subjected to PCR amplification through a literature primer and the innovative primer, the capillary electrophoresis effect comparison shows that the innovative primer is more sensitive, and a positive signal can be detected after 5 ng.
Example 4
In this example, 20 samples, 10 positive patients and 10 negative patients, were tested by using the innovative primer and amplification system of example 1 and the new procedure of example 2, and the capillary electrophoresis effect graphs obtained are shown in the following FIG. 5 and the following FIG. 6, respectively. The result shows that the method disclosed by the invention can be used for rapidly and accurately detecting the sample with the high GGC repetition number.
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.
Reference documents:
1. Tian, Y., et al., Expansion of Human-Specific GGC Repeat in Neuronal Intranuclear Inclusion Disease-Related Disorders. Am J Hum Genet, 2019. 105(1): p. 166-176.
2. Sone, J., et al., Long-read sequencing identifies GGC repeat expansions in NOTCH2NLC associated with neuronal intranuclear inclusion disease. Nat Genet, 2019. 51(8): p. 1215-1221.
sequence listing
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<120> rapid sensitive detection method of NOTCH2NLC gene GGC repetitive sequence and application thereof
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
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gcctgtgctt cggaccgtag cgccag 26
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<213> Artificial Sequence (Artificial Sequence)
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cgcacgctcc tgcacagcct ctcctccgcc gccgccgcc 39
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
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cgcacgctcc tgcacagcct c 21

Claims (8)

1. A primer composition for detecting a GGC repetitive sequence of a NOTCH2NLC gene, which is characterized by comprising: the nucleotide sequence is shown as SEQ ID NO: 1, and the nucleotide sequence of the upstream primer is shown as SEQ ID NO: 2, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 3, or a third primer.
2. An amplification reaction system for amplifying a GGC repeat sequence of a NOTCH2NLC gene, the amplification reaction system comprising the primer composition of claim 1.
3. A kit for rapidly detecting the GGC repeat sequence of a NOTCH2NLC gene, comprising the primer composition of claim 1 or the amplification reaction system of claim 2.
4. A method for rapidly detecting the GGC repeat sequence of NOTCH2NLC gene using the amplification reaction system of claim 2 or the kit of claim 3, wherein the PCR reaction procedure for amplifying the GGC repeat sequence of NOTCH2NLC gene is as follows:
reacting for 10min at the temperature of 98 ℃; then reacting at 98 ℃ for 30sec, at 66 ℃ for 1min and at 68 ℃ for 1min for 20 cycles in total; then reacting at 98 ℃ for 30sec, at 56 ℃ for 30sec, and at 68 ℃ for 1min, and totaling 15 cycles; finally 68 ℃ for 10 minutes.
5. The method of claim 4, comprising:
obtaining genome DNA of a sample to be detected;
PCR amplifying a NOTCH2NLC gene using the amplification reaction system according to claim 2 or the kit according to claim 3;
and detecting the repeated sequence GGC of the NOTCH2NLC gene in the obtained amplification product.
6. The method as claimed in claim 5, wherein the amplification product obtained is detected by capillary electrophoresis for the repeat sequence GGC of NOTCH2NLC gene.
7. Use of the primer composition according to claim 1, the amplification reaction system according to claim 2, the kit according to claim 3 or the method according to claims 4-6 in the medical field.
8. The use of the primer composition of claim 7, the amplification reaction system of claim 2, the kit of claim 3, or the methods of claims 4-6 for aiding in the prediction and/or diagnosis of neuronal endonuclear inclusion diseases.
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115029434A (en) * 2022-07-22 2022-09-09 北京阅微基因技术股份有限公司 NOTCH2NLC gene CGG repetition number detection system and kit

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110577987A (en) * 2019-06-24 2019-12-17 胜亚生物科技(厦门)有限公司 Detection method of CGG (glutamic acid G) repetitive sequence of FMR1 gene and application thereof
CN112094893A (en) * 2019-06-17 2020-12-18 杭州方夏生物科技有限公司 Neuron endonucleosome disease NOTCH2NLC gene GGC repetitive amplification detection kit
CN112553299A (en) * 2019-09-10 2021-03-26 北京大学第一医院 NOTCH2NLC gene GGC repetitive sequence amplification method

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112094893A (en) * 2019-06-17 2020-12-18 杭州方夏生物科技有限公司 Neuron endonucleosome disease NOTCH2NLC gene GGC repetitive amplification detection kit
CN110577987A (en) * 2019-06-24 2019-12-17 胜亚生物科技(厦门)有限公司 Detection method of CGG (glutamic acid G) repetitive sequence of FMR1 gene and application thereof
CN112553299A (en) * 2019-09-10 2021-03-26 北京大学第一医院 NOTCH2NLC gene GGC repetitive sequence amplification method

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115029434A (en) * 2022-07-22 2022-09-09 北京阅微基因技术股份有限公司 NOTCH2NLC gene CGG repetition number detection system and kit
CN115029434B (en) * 2022-07-22 2023-03-17 北京阅微基因技术股份有限公司 NOTCH2NLC gene CGG repetition number detection system and kit

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