Primer, probe and kit for detecting 2385 polymorphism of LRRK2 gene

文档序号：527202 发布日期：2021-06-01 浏览：3次中文

阅读说明：本技术 一种检测lrrk2基因2385多态性的引物和探针及试剂盒 (Primer, probe and kit for detecting 2385 polymorphism of LRRK2 gene ) 是由李振韬郭力榕马雨薇黄俊清于 2021-03-16 设计创作，主要内容包括：本发明公开了一种检测LRRK2基因2385位点多态性的引物和探针及试剂盒。利用1对LRRK2基因2385位点特异性引物,扩增人类LRRK2基因2385位点基因片段,设计特异性Taqman MGB探针,即在一个反应体系中通过两种不同通道检测一个SNP位点。在反应体系含有不同基因型模板的情况下,随着PCR扩增过程,释放不同的荧光信号。利用仪器对PCR过程中相应通道的信号强度进行实时监测和输出,实现检测结果的定性分析。本发明在保证检出特异性和灵敏度的基础之上,实现便捷、快速、高效地检测,减少检测操作步骤、缩减检测反应时间的同时减低了生产成本和检测成本,有利于临床检测分析应用。(The invention discloses a primer, a probe and a kit for detecting 2385 site polymorphism of LRRK2 gene. A human LRRK2 gene 2385 site gene fragment is amplified by utilizing 1 pair of LRRK2 gene 2385 site specific primers, and a specific Taqman MGB probe is designed, namely, one SNP site is detected through two different channels in one reaction system. In the case of a reaction system containing different genotype templates, different fluorescent signals are released along with the PCR amplification process. And (3) utilizing an instrument to carry out real-time monitoring and output on the signal intensity of the corresponding channel in the PCR process, and realizing qualitative analysis of a detection result. On the basis of ensuring the detection specificity and sensitivity, the invention realizes convenient, rapid and efficient detection, reduces the detection operation steps, shortens the detection reaction time, reduces the production cost and the detection cost, and is beneficial to clinical detection analysis application.)

1. A primer and a probe for detecting 2385 polymorphism of LRRK2 gene are characterized by comprising ARMS primers and probes for detecting 2385 site of LRRK2 gene;

the ARMS primer pair and the MGB probe for detecting the LRRK 22385 locus comprise:

2385A site: 5'-AAAGGGCACGCAGTCTATTAGTCT-3'

2385G site: 5'-AAGGGCACGCAGTCTATTAGTCC-3'

2385 downstream general primer: 5'-GCAGCTTTCAGTGATTCC-3'

2385A Probe 5'-AAAAACTCTGTAGCCTA-3'

2385G Probe 5'-AAAAACTCTGTGTGCTA-3'

The 2385A probe is a Taqman probe, a 5 'end is modified with a fluorescent group, and a 3' end is modified with a quenching group;

the 2385G probe is a Taqman probe, and a 5' end is modified with a fluorescent group; and 3' end is modified with quenching group.

2. A kit for detecting 2385 polymorphism of LRRK2 gene, which comprises the primer and probe for detecting 2385 polymorphism of LRRK2 gene of claim 1.

3. The kit for detecting the 2385 polymorphism of the LRRK2 gene, according to claim 2, further comprising an internal control primer pair and a probe for internal quality control:

internal reference upstream primer 5'-ACTGTGGCGTGATGGCCG-3'

Internal reference downstream primer 5'-CTCCGACGCCTGCTTCACCAC-3'

Internal reference probe 5'-TGAACGGGAAGCTCACTGGCAT-3'

The internal reference probe is a Taqman probe, a 5 'end is modified with a fluorescent group, and a 3' end is modified with a quenching group.

4. The kit for detecting 2385 polymorphism of LRRK2 gene according to claim 2, further comprising a PCR premix with anti-PCR enzyme inhibitor, wherein the premix comprises modified Hotstart-Taq polymerase, UNG enzyme, MUSTANG PURPLE fluorescent calibration dye, dNTP, MgCl and dNTP₂、PCR buffer。

5. The kit for detecting the 2385 polymorphism of LRRK2 gene according to claim 2, further comprising a positive control and a negative control, wherein the positive control is a mixture of plasmid DNA of LRRK2 gene 2385A, 2385G gene and plasmid DNA of internal reference; the negative control was ultrapure water containing no target gene and no reference gene.

Technical Field

The invention relates to a primer, a probe and a kit for detecting 2385 polymorphism of LRRK2 gene, belonging to the technical field of gene polymorphism detection.

Background

The human LRRK2 gene is located on chromosome 12p 11.2-q 13.1, is about 144kb (containing 7581 base pairs), contains 51 exons, encodes a protein consisting of 2527 amino acids (molecular weight 275KDa), and belongs to the ROCO protein family, wherein the LRRK2 protein (also called dardardardardrin protein or leucine-rich repeat kinase 2).

Parkinson's Disease (PD) is currently the second most progressive neurodegenerative disease in the world. Certain mutations and risk variations of the leucine-rich repeat kinase 2(LRRK2) gene are very common in PD patients, accounting for approximately 5-15% of familial and sporadic PD. To date, nearly 100 variants of LRRK2 have been discovered. Among them, G2019S, R1628P, and G2385R have conventionally received a great deal of attention. Each of these variants has a different geographical distribution. G2019S is the most common variation in the european/western population, accounting for 3% of familial PD cases in the european population, nearly 14% in the german jewish (AJ), and very rarely in the asian population. G2385R, R1628P and a2385V are more common in the east asian population, with two mutations G2385R, R1628P accounting for 5-9% of asian parkinson's disease population, and both mutations increasing the risk of parkinson's disease 2-fold. And A2385V may indicate the risk of early onset Parkinson's disease in Asian populations.

Parkinson patients carrying the LRRK 2G 2385R mutation have more family history of Parkinson disease, the disease course is longer, and the MMSE score is higher than that of non-carriers. The G2385R variation is found not only in 10% of PD cases, but also in 3-4% of non-diseased populations. The LRRK2 gene Gly2385Arg mutation site is only found in Asian population. The site is located in WD40 structural domain of 48 th exon of LRRK2 gene, so that the 7153 th nucleotide G is changed into A, and the corresponding codon GGG is changed into GGA. When Gly2385Arg is changed, arginine with hydrophilicity and positive charge replaces hydrophobic glycine, so that the surface charge and the conformation of a WD40 structural domain are changed, new interaction with other proteins is generated, the interaction with the original glycine-binding protein is prevented, and the functional performance of LRRK2 protein is influenced finally. At present, a plurality of researches find that the LRRK2 gene Gly2385Arg site is related to sporadic Parkinson disease.

The prodromal phase of PD refers to symptoms or signs of PD neurodegeneration, not accompanied by typical motor parkinsonism. Genetic findings, combined with age, environmental risk factors and other diagnostic marker detection, play an important role in the diagnosis of prodromal PD. LRRK 2G 2385R is a risk factor in asian populations, including chinese and non-chinese. This may help to further explore the etiology and biomarkers of parkinson's disease, particularly in asian populations.

The prior art has the following defects and shortcomings:

the existing no-market kit on the market detects the virus; the ARMS primer and the MGB probe are screened by the kit specificity, the specificity is very strong, the result interpretation does not need to be calculated, and the kit is clear and definite.

In the prior art, detection can only be performed by a sequencing method, machines and consumables required by sequencing are expensive, requirements on quality of operators are high, a common hospital almost has no sequencing service, and screening of gene loci is not facilitated, and a fluorescent quantitative PCR method is used as a gene detection reagent, so that the fluorescent quantitative PCR method can be compatible with almost all fluorescent quantitative PCR instruments at present, and the fluorescent quantitative PCR technology plays an irreplaceable role in new coronary epidemic situations, becomes a mainstream of the detection industry, and has a very wide application prospect.

In the clinical operation level, currently, related gene sequencing has no special charging standard, and because high-throughput sequencing is suitable for simultaneous detection of a plurality of loci and the cost for detecting one gene locus alone is high, generally, charging is carried out according to a plurality of loci, and the economic burden of patients is high. This is not the case with our fluorescent quantitative PCR detection reagent, and one site is charged for the price of one site.

The existing hospital sequencing is generally entrusted to a third-party medical inspection company, the inspection period is long, and the PCR fluorescence detection kit can obtain a result within two hours at the fastest speed, so that the back-and-forth transportation cost of a patient is saved, and a doctor can be helped to perform auxiliary diagnosis on the patient in time.

Disclosure of Invention

The invention aims to provide a detection kit for 2385 site polymorphism of LRRK2 gene, which overcomes the defects in the prior detection technology, realizes rapid, high-flux and low-cost detection of 2385 site polymorphism of LRRK2 gene, and has high sensitivity and strong specificity.

The kit provided by the invention utilizes 1 pair of leucine repeat kinase 2(LRRK2) gene 2385 specific primers to amplify human LRRK2 gene 2385 site gene fragments, and designs specific Taqman MGB probes (5 'end labeled FAM fluorescein and 5' end labeled VIC fluorescein), namely, one SNP site is detected through two different channels in one reaction system. In the case of a reaction system containing different genotype templates, different fluorescent signals are released along with the PCR amplification process. And (3) utilizing an instrument to carry out real-time monitoring and output on the signal intensity of the corresponding channel in the PCR process, and realizing qualitative analysis of a detection result.

The kit provided by the invention is provided with UNG enzyme + dUTP anti-pollution measures, namely, the UNG enzyme is utilized to fully degrade possible PCR product pollution during PCR amplification. To exclude false positive results that may result therefrom.

The kit of the invention uses human GAPDH gene as reference gene to design specific primers and probes (5' end is marked with ROX fluorescein), and detects the working condition of the system in real time, thereby eliminating false negative results.

The invention provides a primer and a probe for detecting 2385 polymorphism of LRRK2 gene, which comprises ARMS primer and probe for detecting 2385 site of LRRK2 gene;

the ARMS primer pair and the MGB probe for detecting the LRRK 22385 locus comprise:

2385A site: 5'-AAAGGGCACGCAGTCTATTAGTCT-3' (SEQ ID NO.1)

2385G site: 5'-AAGGGCACGCAGTCTATTAGTCC-3' (SEQ ID NO.2)

2385 downstream general primer: 5'-GCAGCTTTCAGTGATTCC-3' (SEQ ID NO.3)

2385A Probe 5 'VIC-AAAAACTCTGTAGCCTA-3' MGB (SEQ ID NO.4)

2385G Probe 5 'FAM-AAAAACTCTGTGTGCTA-3' MGB (SEQ ID NO.5)

The kit for detecting the polymorphism of the LRRK2 gene 2385 comprises the primer and the probe for detecting the polymorphism of the LRRK2 gene 2385.

Preferably, the kit also comprises an internal control primer pair and a probe for detecting internal quality control:

internal reference upstream primer 5'-ACTGTGGCGTGATGGCCG-3' (SEQ ID NO.6)

Internal reference downstream primer 5'-CTCCGACGCCTGCTTCACCAC-3' (SEQ ID NO.7)

Internal reference probe 5 'ROX-TGAACGGGAAGCTCACTGGCAT-3' BHQ2(SEQ ID NO.8)

The two different fluorophores of the probe comprise any combination of two fluorophores with different emission wavelengths. Commonly used fluorescent groups include, but are not limited to, available fluorescent labels of various kinds, such as FAM, HEX, VIC, JOE, TET, ALEX-30, ROX, TEXAS RED, CY3, CY5, CY5.5, and the like. Commonly used quenching groups include, but are not limited to, various quenchers currently used, such as Dabcyl, BHQ1, BHQ2, BHQ3, TAMRA, Eclipe, NFQ-MGB, and the like.

The fluorescent probe comprises at least one of a TaqMan probe, a TaqMan-MGB probe, a molecular beacon, a modified molecular beacon, a double-stranded fluorescent substitution probe, a Light Cycler probe, a double-ring probe and the like.

Preferably, the kit further comprises a PCR premix with an anti-PCR enzyme inhibitor, wherein the premix comprises modified Hotstart-Taq polymerase, UNG enzyme, MUSTANG PURPLE fluorescent calibration dye, dNTP, MgCl₂、PCR buffer。

Preferably, the kit also comprises a positive control substance and a negative control substance, wherein the positive control substance is mixed plasmid DNA of LRRK2 gene 2385A and 2385G gene and internal reference plasmid DNA; the negative control was ultrapure water containing no target gene and no reference gene.

The gene polymorphism of the invention comprises 2385AA type, 2385GG type and 2385GA type of LRRK2 gene.

The invention achieves the following beneficial effects:

1. the specificity is high: the kit adopts ARMS primers and MGB probes to specifically detect gene polymorphism.

2. The sensitivity is high: the genotype of 1ng of genomic DNA can be accurately detected.

3. Safe and non-invasive: the sample can be a blood sample, and can be used for collecting human oral epithelial cells conveniently and quickly.

4. Multiple reactions: each reaction system carries out multiple reactions to detect 2 polymorphisms at the same site, thereby reducing the detection operation steps, lowering the cost, improving the detection efficiency and being beneficial to clinical detection analysis application.

And 5, a UNG enzyme anti-pollution system can effectively degrade the PCR product under the room temperature condition, the additional program time is not required to be increased, and the detection time is shortened.

6. The result interpretation does not need calculation, is convenient and intuitive, and has low requirements on operators.

On the basis of ensuring the detection specificity and sensitivity, the invention realizes convenient, rapid and efficient detection, reduces the detection operation steps, shortens the detection reaction time, reduces the production cost and the detection cost, and is beneficial to clinical detection analysis application.

Drawings

FIG. 1 is a graph showing the result of detection of a wild-type sample LRRK 22385G/G;

FIG. 2 is a graph showing the results of detection of LRRK 22385A/A homozygous mutant samples;

FIG. 3 is a graph showing the results of detection of LRRK 22385G/A heterozygous mutant samples.

Detailed Description

The present invention will be further described with reference to the following examples. The following examples are only for illustrating the technical solutions of the present invention more clearly, and the protection scope of the present invention is not limited thereby.

Example (b):

1. the main components of the kit

2. Reagent preparation

2.1 the PCR reaction mixture (at position 2385) was removed, thawed at room temperature (25 ℃), mixed by shaking for 30 seconds, and centrifuged at 2000rpm for 10 seconds.

2.2 the number of reactions (n) required for the current experiment was counted, and each PCR reaction solution was dispensed into n reaction tubes at a dispensing rate of 16. mu.L/well. Different systems need to be marked, so that confusion is avoided. The PCR reaction tube is transferred to a sample preparation area, and the rest reagents are put back to a refrigerator at the temperature lower than-15 ℃ for freezing and storing in dark place.

number of samples + negative control (1 part of a person) + positive control (1 part of a person)

3. Sample application

3.1 adding the genome DNA of the sample to be detected, the negative control and the positive control into the reaction tube filled with the PCR reaction solution respectively, wherein the adding amount of the DNA is 4 mu L/hole.

3.2 cover the PCR reaction tube, and record the sample loading condition. Centrifuging the PCR amplification tube at 4000rpm for 5min to avoid tube wall hanging beads; and transferring the PCR reaction tube to a nucleic acid amplification area for on-machine detection. And if the template cannot be immediately installed in the PCR reaction tube when meeting the temporary condition after being added with the template, the PCR reaction tube with the template added is recommended to be temporarily stored at the temperature of 2-8 ℃, and is installed in the machine for detection as soon as possible within 2 hours.

4. PCR amplification (performed in the amplification and analysis area, please refer to the instructions for each instrument)

4.1 starting up and carrying out instrument performance self-checking.

4.2 taking the PCR reaction tube added with the DNA to be detected, placing the PCR reaction tube at a corresponding position of a sample groove of the instrument, and recording the placing sequence.

4.3 cycle parameter settings (Protocol):

4.4 run the reaction program.

5. Analysis of results

And after the reaction is finished, analyzing the amplification curve and the curve of the corresponding internal reference respectively. Adjusting a Begin value and an End value of the Baseline threshold according to the analyzed image (a user can adjust the Begin value by himself according to the actual situation, the Begin value can be set to be 2-15, the End value can be set to be 5-25, the amplification curve of the negative control product is adjusted to be straight or lower than a threshold line), the amplification curve is made to just exceed the highest point of the normal negative control product, and then the quantitative result is recorded.

And (3) judging the validity of the detection result:

(1) negative control: FAM and VIC channels have no amplification curve, or the amplification curve is a straight line or a slight oblique line, and has no obvious exponential growth period, and has no Ct (Cq) value or a detection Ct (Cq) value of more than 35;

(2) positive control: the Ct (Cq) value of FAM and VIC channels is less than or equal to 35, and the amplification curve has an obvious exponential amplification period.

The above requirements need to be met simultaneously in the same experiment, otherwise, the experiment is invalid, and errors in instruments, reagents, amplification conditions and the like need to be checked.

And (3) judging the validity of the sample:

(1) internal reference gene: the Ct value of the ROX channel in all sample detections is less than 35, and the amplification curve has obvious exponential growth period.

(2) As shown in fig. 1 to 3, determination of the detection result:

compared with the prior art, the invention has the advantages that:

in the prior art, the specificity is not strong, the difference between the wild type and the mutant type is not obvious enough, the judgment is carried out by adopting a delta Ct and combining an amplification curve, the result judgment is complicated, and the requirement on a judgment personnel is high; the kit has the advantages of strong specificity, no need of calculation for result interpretation, and clearness in screening the ARMS primer and the MGB probe.

In the prior art, the requirement on sample processing is high when the 2385 site polymorphism of LRRK2 gene is detected, and blood samples are mostly needed; the kit sample of present case not only can adopt the blood sample can also gather human oral epithelial cell, need not the blood sampling, and safe noninvasive, convenient and fast.

In the prior art, each detection system can only carry out single PCR reaction for each detection, and each reaction system can only detect the defect of one gene locus polymorphism; the gene detection reagent is prepared by premixing a plurality of pairs of primers, a plurality of specific fluorescent probes modified by different fluorescent dye groups and PCR premix solution to form a reaction system, so that different target genes are efficiently amplified by multiplex PCR under the combination of the plurality of pairs of specific primers, and MGB-NFQ and TaqMan-BHQ fluorescent probes for marking different fluorescent dye groups are used for detecting and analyzing target products in real time.

The PCR anti-pollution system in the prior art needs additional program time, and the gene detection reagent UNG enzyme anti-pollution system can effectively degrade PCR products under room temperature conditions, does not need additional program time, and shortens detection time.

The above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, several modifications and variations can be made without departing from the technical principle of the present invention, and these modifications and variations should also be regarded as the protection scope of the present invention.

Sequence listing

<110> Jiangsu Begel biopharmaceutical GmbH

<120> primer, probe and kit for detecting 2385 polymorphism of LRRK2 gene

<141> 2021-03-16

<160> 8

<170> SIPOSequenceListing 1.0

<210> 1

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<212> DNA