CN112608991A - qMS-PCR kit for detecting Prader-Willi syndrome/Angelman syndrome - Google Patents
qMS-PCR kit for detecting Prader-Willi syndrome/Angelman syndrome Download PDFInfo
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Abstract
The invention discloses an qMS-PCR kit for detecting Prader-Willi syndrome/Angelman syndrome. The kit comprises a primer pair and a TaqMan-MGB probe for detecting the methylation of the SNRPN promoter region, and a primer pair and a TaqMan-MGB probe for detecting the non-methylation of the SNRPN promoter region; the 5' end of the upstream primer in the primer pair is connected with a self-closed sequence which can form a stem-loop structure with a PCR target fragment. When the methylation primer with the unique self-complementary sequence arranged at the 5' end is combined with the non-specificity to the non-methylation sequence template, the sequence can be completely combined with the non-methylation sequence on the template and extends automatically, so that the non-specific amplification is blocked, the amplification efficiency of the methylation conversion sequence is greatly improved, and the generated taqman probe hydrolysis signal Ct value can more accurately judge the methylation state of the SNRPN.
Description
Technical Field
The invention belongs to the field of medical detection, and particularly relates to an qMS-PCR kit for detecting Prader-Willi syndrome/Angelman syndrome.
Background
The Prader-Willi syndrome (PWS) and Angelman Syndrome (AS) are two clinically distinct neurogenetic diseases. PWS (MIM176270) is characterized by: fetal movement reduction, obesity, dystonia in infancy, mental retardation, short stature, hypogonadism due to hypogonadotropic hormone secretion, and abnormal hands and feet. AS (MIM105830) is characterized by severe movement, intellectual disability, ataxia, low muscle tone, epilepsy, speech disorders and a special look characterized by massive mandible and mouth opening for tongue vomiting. Both PWS and AS are caused by the functional defect of the imprinted gene of the chromosome 15q11-13 region, the incidence rate is about 1/10000-1/30000, and the PWS and AS are the genetic imprinted diseases with the highest incidence rate at present. PWS is one of the important causes of symptomatic morbid obesity, and early diagnosis and reasonable intervention are of great importance to improve the quality of life of children patients and prevent serious complications.
PWS/AS pathogenesis is represented by a number of molecular defect classes, including:
1. deletion (c): the deletion of the 15q11-13 region is the main molecular pathological type of the disease of PWS and AS patients, PWS is the deletion of the parent source chromosome 15q11-13, and AS is the deletion of the parent source chromosome 15q 11-13. The western population has 65-75% of patients with deletion type, and the Chinese and Asian population has a higher proportion of the deletion type, more than 80%.
2. Uniparental disomy (UPD): both chromosomes 15 of PWS and AS patients are normal, but both chromosomes 15 of PWS patients are from the mother, i.e., the mother's uniparental disomy (UPD); whereas, both chromosomes 15 of AS patients are from the father, the father uniparental disomy (UPD). The UPD incidence of PWS is more prevalent (20-30%) and the AS is lower (2%).
3. Imprinting mutation: microdeletion and mutation (1-3%) of the Center of Imprinting (IC).
For different types of molecular defect forms, the current genetic diagnosis methods developed at home and abroad for PWS/AS syndrome include high resolution chromosome karyotype analysis (HRB), Fluorescence In Situ Hybridization (FISH), microsatellite linkage analysis, methylation analysis and the like. HRB was the first cytogenetic test method to be applied in PWS/AS studies. The method can only diagnose the deletion type PWS/AS, and can not detect all the deletions, and the detection rate is about 60 percent. FISH is a nonradioactive in situ hybridization method, and can diagnose patients with tiny deletion in the 15q11-13 region or translocation of the 15q related region, which is difficult to find by applying the conventional chromosome banding technology, so that the detection rate of the disease is improved to 70-75%. Microsatellite linkage analysis, such as Short Tandom Repeat (STR) genetic linkage analysis, is identified by comparing the number of repeats or length of each STR locus tested between parental and progeny individuals and by virtue of the fact that the PWS caused by the UPD still has the loss of parent/maternal information outside the 15q11-13 region. The imprinting hybridization (Southern Blotting) is based on the principle that the methylation modification mode of the imprinted gene in the 15q11-13 region is different, wherein the gene is the most common candidate gene for PWS gene mutation research by using the method at present. The method has strong specificity and reliable diagnosis result. However, the method is complicated, needs to use isotope labeling and is difficult to be widely applied.
The SNRPN gene promoter region contains CpG islands where the parental allele is methylated and the maternal allele is unmethylated. Only methylated alleles exist in PWS patients, but only unmethylated alleles exist in AS patients, so that MS-PCR can be used for detecting the methylation state of CpG islands of SNRPN genes, and PWS and AS can be rapidly diagnosed. The principle is as follows: chemical agents such as bisulfite convert all unmethylated cytosines to uracil, whereas cytosines in the CpG of the SNRPN gene are unchanged by methylation. Based on the difference, designing methylation specific primers can distinguish a DNA sequence chemically modified by a methylation allele from a non-methylation allele (as shown in figure 1), adding a specific fluorescent probe targeting the methylation sequence into a PCR reaction system, wherein the probe is only specifically combined with a methylation template, the combination site of the probe is positioned between the two primers, and monitoring the whole PCR process by real-time quantitative PCR by using fluorescent signal accumulation. The methylation state of SNRPN can be accurately judged, and the rapid and effective gene diagnosis and prenatal diagnosis can be carried out on almost all PWS and about 80 percent AS patients.
qMS-PCR detection of PWS/AS is not limited to the molecular defect type of its pathogenesis, and has the capability of efficiently detecting PWS/AS patients, however qMS-PCR is still not applied on a large scale on the market at present, which is mainly determined by the technical characteristics of qMS-PCR. qMS-PCR detection quality is highly limited by bisulfite conversion efficiency, if bisulfite conversion efficiency of cytosine to uracil is too low, then unmethylated CpG sites are easily misinterpreted as methylated CpG sites in qPCR reaction, resulting in false positives; meanwhile, DNA treated by bisulfite is seriously fragmented, the diversity of DNA sequences is reduced after cytosine is converted into uracil, a complex secondary structure is more easily formed, the efficiency of qMS-PCR is further influenced, and the difficulty limits the clinical application of qMS-PCR.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide an qMS-PCR kit for detecting Prader-Willi syndrome/Angelman syndrome.
Another object of the present invention is to provide the use of the qMS-PCR kit for detecting Prader-Willi syndrome/Angelman syndrome as described above.
The purpose of the invention is realized by the following technical scheme:
an qMS-PCR kit for detecting Prader-Willi syndrome/Angelman syndrome, which comprises a primer pair for detecting methylation of a SNRPN promoter region, a primer pair for detecting non-methylation of the SNRPN promoter region, a TaqMan-MGB probe for detecting methylation of the SNRPN promoter region and a TaqMan-MGB probe for detecting non-methylation of the SNRPN promoter region; wherein, the 5 'end of the upstream primer in the primer pair for detecting the methylation of the SNRPN promoter region is connected with a 5' self-blocking sequence which can form a stem-loop structure with a PCR target fragment, and the 5 'end of the upstream primer in the primer pair for detecting the non-methylation of the SNRPN promoter region is connected with a 5' self-blocking sequence which can form a stem-loop structure with a PCR target fragment.
The 5' self-blocking sequence is designed based on the fact that the complementary fragment contains as many methylation sites as possible.
The length of the 5' self-closed sequence is preferably 10-30 bp; preferably 15-25 bp; more preferably 15 to 20 bp.
The fluorescent label modified on the TaqMan-MGB probe for detecting the methylation of the SNRPN promoter region is different from the fluorescent label modified on the TaqMan-MGB probe for detecting the non-methylation of the SNRPN promoter region.
The fluorescent marker modified on the TaqMan-MGB probe for detecting the methylation of the SNRPN promoter region is preferably VIC.
The fluorescence label modified on the TaqMan-MGB probe for detecting the non-methylation of the SNRPN promoter region is preferably FAM.
The primer pair for detecting the methylation of the SNRPN promoter region is preferably as follows:
SNRPN-M-F：5’-AACCACACAAACATACT GAGTTGGGATTTTTGTATTG-3’；
SNRPN-M-R：5’-ACGCCAAACTCGCTACAACAACGA-3’。
the primer pair for detecting the non-methylation of the SNRPN promoter region is preferably as follows:
SNRPN-N-F：5’-GACCGCGCAAACGTACT GAGTTGGGATTTTTGTATTG-3’；
SNRPN-N-R：5’-ACACCAAACTCACTACAACAACAA-3’。
the TAQMAN-MGB probe for detecting the methylation of the SNRPN promoter region is preferably as follows:
SNRPN-M-P：5’-VIC-AGGTTGGCGCGTATGTTTA-MGB-3’。
the TAQMAN-MGB probe for detecting the non-methylation of the SNRPN promoter region is preferably as follows:
SNRPN-N-P：5’-FAM-AGGTTGGTGTGTATGTTTA-MGB-3’。
the kit also comprises a TaqMan PCR reagent.
The qMS-PCR kit for detecting Prader-Willi syndrome/Angelman syndrome is applied to the detection of Prader-Willi syndrome/Angelman syndrome in non-diagnostic purposes, and preferably comprises the following steps:
(1) carrying out bisulfite conversion on the genome DNA of a sample to be detected to obtain converted genome DNA;
(2) using the transformed genome DNA as a template, and applying the qMS-PCR kit for detecting Prader-Willi syndrome/Angelman syndrome to carry out TaqMan PCR amplification;
(3) interpretation of the results:
A. the SNRPN methylated probe signal is + and the SNRPN unmethylated probe signal is + and is a normal person;
B. the SNRPN methylated probe signal is + and the SNRPN unmethylated probe signal is-is the PWS patient;
C. SNRPN methylated probe signal is-SNRPN unmethylated probe signal is + and AS patients are identified.
The non-diagnostic purpose may be for experimental research purposes.
The sample to be tested in the step (1) is preferably peripheral blood.
The optimized condition of TaqMan PCR amplification in the step (2) is 95 ℃ for 10 min; at 95 ℃ for 15s and 60 ℃ for 1min for 40 cycles.
The results described in step (3) are preferably obtained by SDS software analysis.
Compared with the prior art, the invention has the following advantages and effects:
the invention uses qMS-PCR (Quantitative Methylation-Specific real-time PCR) technology to qualitatively and quantitatively detect the Methylation state of the promoter region of the SNRPN gene.
The inventor of the invention firstly applies a 5 'self-closed primer setting mode, sets a unique self-complementary sequence at the 5' end of a methylated primer, wherein the sequence has a complementary sequence, when the methylated primer is combined with a non-specificity to a non-methylated sequence template, the sequence can be completely combined with the non-methylated sequence on the template and extends automatically, so as to block non-specific amplification, greatly improve the amplification efficiency of a methylated conversion sequence (as shown in figure 2), generate a taqman probe hydrolysis signal Ct value more ahead, have a stronger signal value, and more accurately judge the methylation state of SNRPN (as shown in figure 3).
Drawings
FIG. 1 is a schematic diagram of qMS-PCR of the prior art; wherein A is a schematic diagram of C base converted and unmethylated by bisulfite; b is a schematic design pattern of qMS-PCR by setting double probes for CpG sites of different methylation states.
FIG. 2 is a schematic diagram of the principle of the present invention for masking non-specific amplification by using 5' self-blocking primer design.
FIG. 3 is a graph showing the results of qMS-PCR using different primers on a sample to be tested for PWS syndrome; wherein, A uses a conventional primer, curve a is a non-specific non-methylation signal, and curve b is a specific methylation signal; b, using the improved 5' self-sealing primer, wherein the curve c is a non-specific non-methylation signal, the curve d is a specific methylation signal, the curve c is basically eliminated, the curve d is enhanced, and the Ct value is advanced by 2-3 cycles.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.
Example 1 design of primers and TaqMan-MGB probes for Prader-Willi syndrome/Angelman syndrome detection
According to the SNRPN gene promoter sequence, a methylated sequence and an unmethylated sequence after bisulfite conversion are obtained by using Methyl Primer Express v1.0 software, a Primer Premier5 software is used for designing a Primer and a TaqMan probe aiming at a methylated and unmethylated difference sequence, and a 5' self-closed sequence is designed at a proper position of an upstream Primer after the Primer is selected so as to be combined with the unmethylated sequence which is not specifically amplified to form a stem-loop structure.
The design positions of the primers and the probes are shown by taking the unmethylated sequences as an example:
the sequences of the primers and the TaqMan-MGB probe are as follows:
(1) primer pair for detecting SNRPN methylation:
SNRPN-M-R：5’-ACGCCAAACTCGCTACAACAACGA-3’。
(2) primer pair for detecting SNRPN non-methylation:
SNRPN-N-R：5’-ACACCAAACTCACTACAACAACAA-3’。
(3) TaqMan-MGB sense probe for detecting SNRPN methylation:
SNRPN-M-P：5’-VIC-AGGTTGGCGCGTATGTTTA-MGB-3’。
(4) TaqMan-MGB probe for detecting SNRPN non-methylation:
SNRPN-N-P：5’-FAM-AGGTTGGTGTGTATGTTTA-MGB-3’。
example 2 bisulfite conversion of genomic DNA
1) Obtaining genomic DNA of a sample
Sample extraction is carried out by using a magnetic bead method DNA extraction kit of Guangzhou Secho biological science and technology GmbH, adding 200 mu L of peripheral blood, adding 200 mu L of lysate and 10 mu L of proteinase K, and carrying out constant temperature shaking lysis at 56 ℃ for 10 min; adding 300 mu L of isopropanol and 15 mu L of magnetic beads after instantaneous centrifugation, uniformly mixing, placing on a magnetic frame for adsorption for 3min, and discarding the supernatant; adding a washing solution VW 1400 mu L of heavy suspension magnetic beads, uniformly mixing, placing on a magnetic frame for adsorption for 3min, and discarding the supernatant; adding 2400 mu L of washing liquid VW for resuspension magnetic beads, uniformly mixing, placing on a magnetic frame for adsorption for 3min, discarding the supernatant, and repeating the step once; adsorbing and airing on a magnetic frame for 5-8min, adding 100 mu L of eluent, shaking and cracking at the constant temperature of 56 ℃ for 10min, putting the centrifugal tube into the magnetic frame for adsorbing for 5min, and absorbing supernatant, namely the extracted total DNA of the blood.
2) Bisulfite conversion of genomic DNA
The DNA extraction kit of magnetic bead method of Jiangsu Jingshan biotech GmbH is used for sample transformation, a proper amount of melted genome DNA is taken out to be a 2.0ml microcentrifuge tube, then an eluent (Elution buffer) is added to be 100 mu l, and the mixture is vortexed, mixed and centrifuged for a short time. Adding 150 mul of sulfite solution and 25 mul of protective solution into a centrifuge tube; covering the microcentrifuge tube tightly, mixing sulfite reaction liquid uniformly in a vortex mode, and centrifuging the microcentrifuge tube for a short time; placing the microcentrifuge tube in a constant-temperature oscillation incubator, and incubating for 45 +/-5 minutes at a constant temperature of 80 ℃ without oscillation; after incubation for 45 +/-5 minutes, immediately taking out the microcentrifuge tube;
adjusting the temperature of the constant-temperature oscillation incubator to 23 +/-2 ℃ for subsequent use; temporarily centrifuging the micro-centrifuge tube, adding 1000. mu.l of washing solution A and 20. mu.l of uniformly mixed (fresh and resuspended) magnetic beads, uniformly mixing by vortex, placing in a constant-temperature oscillation incubator at 23 +/-2 ℃, and incubating for 45 +/-5 min; after short-time centrifugation, placing the mixture in a magnetic test tube rack for adsorption for 3min, and discarding the supernatant; adding 800 mu L of washing solution A for resuspension magnetic beads, uniformly mixing, placing on a magnetic frame for adsorption for 3min, and discarding the supernatant; adding 800 μ L of washing solution A, placing on a magnetic frame, adsorbing for 3min, discarding the supernatant, and repeating the steps once; removing residual liquid with 10-100 μ l gun head, drying for 10min, and drying the precipitate; adding 100 mu L of eluent, shaking at the constant temperature of 56 ℃ for 10min, putting the centrifugal tube into a magnetic frame for adsorption for 5min, and absorbing supernatant to obtain DNA converted from the bisulfite.
Example 3: PCR amplification and result interpretation
1) Using the bisulfite conversion DNA obtained in example 2 as a template, under the guide of the primers and TaqMan-MGB probe described in example 1, PCR amplification was performed by ABI 7500 real-time fluorescence quantitative PCR instrument (applied biosystems, USA), and the amplification was completed and analyzed according to the operation steps of the allele-discriminating experiment, i.e., reading the signal before PCR amplification in the allele-discriminating experiment, executing the amplification procedure, and reading and analyzing the signal after amplification were sequentially performed. Wherein, the PCR reaction system is a 20 mu L reaction system: 10 μ L of 2 × TaqMan universal PCR amplification pre-mixed reagent (purchased from applied biosystems, USA) is added, the final concentration of each reagent in the reaction system is 900nM, the final concentration of the 2 fluorescence labeled TaqMan-MGB probes in the reaction system is 200nM and 150ng DNA. The PCR reaction conditions are as follows: firstly, the temperature is 95 ℃ for 10 min; then, the temperature was increased to 95 ℃ for 15 seconds and 60 ℃ for 1min for 40 cycles.
2) To obtain the result
The results of the experiment were analyzed by SDS software (applied biosystems, USA) to obtain the methylation state of the SNRPN promoter region, and to further determine the risk of PWS/AS of the subjects. A total of 3 samples (from Guangdong) of subjects were taken for this experiment, and the final genotype distributions are shown in Table 1 below.
TABLE 1
| Test subject | SNRPN methylation probe signals | SNRPN unmethylated probe signal | PWS/AS status interpretation |
| 1 | + | + | Is normal |
| 2 | + | - | PWS patients |
| 3 | - | + | AS patients |
Note: SDS software sets Presence/Absence mode, judges whether there is signal directly; + is a signal and-is no signal.
Example 4: comparison of detection effects of 5' self-blocked primer and conventional primer
Samples of 10 PWS syndrome patients and 10 AS syndrome patients are selected in the experiment, the type of the detected sample is dry blood slices commonly used for genetic screening, the dry blood slices are soaked in buffer solution, DNA is extracted and transformed according to the steps, and qMS-PCR detection is carried out. The DNA content in the dried blood slice is less, the requirements on the sensitivity and the specificity of the detection method are higher, 5' self-blocking primers and conventional primers are used for respectively detecting blood slice samples in the experiment, and the sensitivity and the specificity of the detection method are compared. The conventional primer differs from the primer with a 5 'self-blocking primer only in that the upstream primer in the pair of primers for detecting SNRPN methylation and the pair of primers for detecting SNRPN non-methylation does not contain a box-out portion, i.e., does not contain a 5' self-blocking sequence. The comparison results are shown in table 2. As is clear from the results in Table 2, in 10 cases of PWS patients, the qMS-PCR reaction with the 5' self-blocking primer enabled accurate detection of SNRPN promoter methylation and accurate judgment of PWS/AS; the effect of the common primers is poor, 1 of 10 samples is judged to be normal by mistake, and 1 sample cannot be judged without signals; the situation was similar in patients with AS, and 1 patient with AS was misjudged to be normal in the ordinary primer test.
TABLE 2
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Sequence listing
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Claims (10)
1. An qMS-PCR kit for detecting Prader-Willi syndrome/Angelman syndrome, characterized in that: the kit comprises a primer pair for detecting the methylation of the SNRPN promoter region, a primer pair for detecting the non-methylation of the SNRPN promoter region, a TaqMan-MGB probe for detecting the methylation of the SNRPN promoter region and a TaqMan-MGB probe for detecting the non-methylation of the SNRPN promoter region; wherein, the 5 'end of the upstream primer in the primer pair for detecting the methylation of the SNRPN promoter region is connected with a 5' self-blocking sequence which can form a stem-loop structure with a PCR target fragment, and the 5 'end of the upstream primer in the primer pair for detecting the non-methylation of the SNRPN promoter region is connected with a 5' self-blocking sequence which can form a stem-loop structure with a PCR target fragment.
2. The qMS-PCR kit for detecting Prader-Willi syndrome/Angelman syndrome according to claim 1, wherein: TaqMan PCR reagents were also included.
3. The qMS-PCR kit for detecting Prader-Willi syndrome/Angelman syndrome according to claim 1 or 2, characterized in that: the 5' self-blocking sequence is designed based on the fact that the complementary fragment contains as many methylation sites as possible.
4. The qMS-PCR kit for detecting Prader-Willi syndrome/Angelman syndrome according to claim 3, wherein: the length of the 5' self-closed sequence is 10-30 bp; further 15-25 bp; further 15-20 bp.
5. The qMS-PCR kit for detecting Prader-Willi syndrome/Angelman syndrome according to claim 1 or 2, characterized in that: the fluorescent label modified on the TaqMan-MGB probe for detecting the methylation of the SNRPN promoter region is different from the fluorescent label modified on the TaqMan-MGB probe for detecting the non-methylation of the SNRPN promoter region.
6. The qMS-PCR kit for detecting Prader-Willi syndrome/Angelman syndrome according to claim 5, wherein:
the modified fluorescent label VIC on the TaqMan-MGB probe for detecting the methylation of the SNRPN promoter region is shown in the specification;
the modified fluorescent label on the TaqMan-MGB probe for detecting the non-methylation of the SNRPN promoter region is FAM.
7. The qMS-PCR kit for detecting Prader-Willi syndrome/Angelman syndrome according to claim 1 or 2, characterized in that: the primer pair for detecting the methylation of the SNRPN promoter region is as follows:
SNRPN-M-F：5’-AACCACACAAACATACT GAGTTGGGATTTTTGTATTG-3’；
SNRPN-M-R：5’-ACGCCAAACTCGCTACAACAACGA-3’；
the primer pair for detecting the non-methylation of the SNRPN promoter region comprises the following steps:
SNRPN-N-F：5’-GACCGCGCAAACGTACT GAGTTGGGATTTTTGTATTG-3’；
SNRPN-N-R：5’-ACACCAAACTCACTACAACAACAA-3’；
the TaqMan-MGB probe for detecting the methylation of the SNRPN promoter region comprises the following components:
SNRPN-M-P：5’-VIC-AGGTTGGCGCGTATGTTTA-MGB-3’；
the TaqMan-MGB probe for detecting the non-methylation of the SNRPN promoter region comprises the following components:
SNRPN-N-P：5’-FAM-AGGTTGGTGTGTATGTTTA-MGB-3’。
8. use of the qMS-PCR kit for detecting Prader-Willi syndrome/Angelman syndrome according to any one of claims 1 to 7 for detecting Prader-Willi syndrome/Angelman syndrome for non-diagnostic purposes.
9. Use according to claim 8, characterized in that it comprises the following steps:
(1) carrying out bisulfite conversion on the genome DNA of a sample to be detected to obtain converted genome DNA;
(2) using the transformed genome DNA as a template, and applying the qMS-PCR kit for detecting Prader-Willi syndrome/Angelman syndrome to carry out TaqMan PCR amplification;
(3) interpretation of the results:
A. the SNRPN methylated probe signal is + and the SNRPN unmethylated probe signal is + and is a normal person;
B. the SNRPN methylated probe signal is + and the SNRPN unmethylated probe signal is-is the PWS patient;
C. SNRPN methylated probe signal is-SNRPN unmethylated probe signal is + and AS patients are identified.
10. Use according to claim 9, characterized in that:
the sample to be detected in the step (1) is peripheral blood;
the TaqMan PCR amplification condition in the step (2) is 95 ℃ for 10 min; at 95 ℃ for 15s and 60 ℃ for 1min for 40 cycles;
the results described in step (3) were obtained by SDS software analysis.
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