CN112831556A - Kit for detecting MYD88L265P mutation based on AS-PCR and application thereof - Google Patents
Kit for detecting MYD88L265P mutation based on AS-PCR and application thereof Download PDFInfo
- Publication number
- CN112831556A CN112831556A CN202110189489.1A CN202110189489A CN112831556A CN 112831556 A CN112831556 A CN 112831556A CN 202110189489 A CN202110189489 A CN 202110189489A CN 112831556 A CN112831556 A CN 112831556A
- Authority
- CN
- China
- Prior art keywords
- primer
- seq
- myd88
- probe
- mutation
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6858—Allele-specific amplification
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Analytical Chemistry (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Physics & Mathematics (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Pathology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to the technical field of gene mutation detection, and particularly relates to a kit for detecting MYD88L265P mutation based on AS-PCR and application thereof. The invention adopts AS-PCR technology, and inhibits non-specific amplification to the maximum extent by optimizing an amplification system, so that the detection sensitivity reaches 75 copies/mu l, and 0.12% of mutation can be detected at the minimum. And the method is simple and convenient to operate, short in report period and suitable for conventional detection.
Description
Technical Field
The invention relates to the technical field of gene mutation detection, and particularly relates to a kit for detecting MYD88L265P mutation based on AS-PCR and application thereof.
Background
MYD88 is a linker molecule for the Toll-like receptor and interleukin-1 receptor signaling pathways. MYD88L265P somatic mutation can cause abnormal activation of NF-kB signal channel, promote cell proliferation and inhibit apoptosis. The incidence of the MYD88L265P mutation in Waldenstrom Macroglobulinemia (WM) is more than 90%, and is an important marker for WM diagnosis and differential diagnosis. MYD88 mutation status is also a prognostic factor for WM. There are studies showing that the prognosis is better for the MYD88L265P mutant. In addition, the novel small-molecule antitumor drug ibrutinib is the first choice for treating recurrence-refractory WM, and the curative effect of ibrutinib is closely related to the MYD88 mutation state. Therefore, the method has great significance for detecting the mutation MYD88L 265P.
The MYD88L265P mutation is a somatic mutation that is only present in tumor cells of patients, but not in normal cells, and therefore, a highly sensitive detection method is needed to enrich a very small amount of mutant target sequence from a large wild-type background and achieve accurate detection. The existing methods for detecting MYD88L265P mainly include one-generation sequencing and allele-specific PCR (AS-PCR). One generation sequencing has limited sensitivity and cannot be quantified. The NCCN guidelines in 2018 clearly indicate AS-PCR AS the recommended detection method.
There are several reports of using AS-PCR to detect the mutation MYD88L 265P. For example: the patent CN110982898A discloses a MyD88 gene L265P mutation detection kit and detection method based on ARMS-PCR, wherein a mismatch is artificially introduced at the 3' end of a specific primer to improve the specificity of the primer, and the concentrations of a DNA template and the primer in the reaction are controlled to be at a lower level to ensure the specificity of the reaction; the lower limit of the mutation rate detectable by this method is 2%. Patent CN111593126A discloses a primer and a probe for detecting MYD88 gene L265P mutation and a high-sensitivity detection method, wherein an AS-PCR fluorescence method is used to match with the primer and the probe obtained by screening, so that the specificity and the sensitivity of detection are improved, and a bone marrow sample with abnormal cell content AS low AS 2.6% can be detected.
Although the AS-PCR is utilized to realize sensitive detection of the MYD88L265P mutation, the detection requirement of the MYD88L265P mutation is higher and higher with the continuous development and treatment progress of new drugs. The detection sensitivity of the existing common AS-PCR technology is about 1 percent at most. When the mutation rate of the sample is further reduced to below 1%, the Ct value detected by using the existing AS-PCR system is close to the Ct value of the negative sample, the system is unstable, and nonspecific amplification exists to a certain extent, so that the weak positive sample and the negative sample are easily confused. Therefore, how to further improve the detection sensitivity of the AS-PCR on the MYD88L265P mutation is the technical difficulty in the field at present.
Disclosure of Invention
Aiming at the prior art, the invention aims to provide a kit for detecting MYD88L265P mutation based on AS-PCR and application thereof. The invention adopts AS-PCR technology, and inhibits non-specific amplification to the maximum extent by optimizing an amplification system, so that the detection sensitivity reaches 75 copies/mu l, and 0.12% of mutation can be detected at the minimum. And the method is simple and convenient to operate, short in report period and suitable for conventional detection.
In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect of the invention, there is provided a primer-probe combination for detecting a MYD88L265P mutation, comprising: a primer-probe combination A for detecting MYD88L265P mutant and a primer-probe combination B for detecting MYD88L265P wild type;
the primer-probe combination A comprises: a MYD88 wild blocking primer shown in SEQ ID NO.1, a MYD88 mutation upstream primer shown in SEQ ID NO.3, a MYD88 downstream primer shown in SEQ ID NO.5 and a MYD88 probe shown in SEQ ID NO. 6;
the primer-probe combination B comprises: MYD88 mutation blocking primer shown in SEQ ID NO.2, MYD88 wild upstream primer shown in SEQ ID NO.4, MYD88 downstream primer shown in SEQ ID NO.5 and MYD88 probe shown in SEQ ID NO. 6.
In a second aspect of the invention, there is provided the use of a primer-probe combination as described above in the preparation of a reagent and/or kit for detecting a mutation in MYD88L 265P.
In a third aspect of the invention, a kit for detecting mutation of MYD88L265P is provided, and the kit contains the primer-probe combination.
Further, the kit further comprises: an NADPH upstream primer shown in SEQ ID NO.7, an NADPH downstream primer shown in SEQ ID NO.8 and an NADPH probe shown in SEQ ID NO. 9.
Further, the kit further comprises: mutant plasmid standards and wild plasmid standards; the nucleotide sequence of the mutant plasmid standard substance is shown as SEQ ID NO.10, and the nucleotide sequence of the wild plasmid standard substance is shown as SEQ ID NO. 11.
Preferably, in the kit, a primer-probe combination A, an NADPH upstream primer shown in SEQ ID NO.7, an NADPH downstream primer shown in SEQ ID NO.8 and an NADPH probe shown in SEQ ID NO.9 are mixed in a PCR reaction tube in advance; the primer-probe combination B, the NADPH upstream primer shown in SEQ ID NO.7, the NADPH downstream primer shown in SEQ ID NO.8, and the NADPH probe shown in SEQ ID NO.9 were mixed in advance in another PCR reaction tube.
In a fourth aspect of the present invention, there is provided use of the primer-probe combination or the kit as described above for predicting the efficacy of a drug for the treatment of macroglobulinemia Fahrenheit.
In a fifth aspect of the invention, there is provided a method for detecting a mutation in MYD88L265P for non-diagnostic purposes, comprising the steps of:
using DNA of a sample to be detected as a template, respectively constructing a wild type amplification system and a mutant type amplification system by adopting the kit, and respectively performing PCR amplification by using the wild type amplification system and the mutant type amplification system; and quantifying the mutation copy number of the sample to be detected by using the mutation plasmid standard substance, quantifying the wild copy number of the sample to be detected by using the wild plasmid standard substance, and calculating to obtain the mutation rate of MYD88L 265P.
Preferably, the wild-type amplification system comprises: MYD88 wild upstream primer 0.8 mu L, MYD88 downstream primer 0.8 mu L, MYD88 Probe 0.4 mu L, MYD88 mutation blocking primer 0.8 mu L, NAPDH upstream primer 0.3 mu L, NAPDH downstream primer 0.3 mu L, NADPH Probe 0.2 mu L, AceQ U + Probe Master Mix 12 mu L, ddH2O1.2. mu.L, template 5. mu.L.
The mutant amplification system comprises: MYD88 mutation upstream primer 0.8 mu L, MYD88 downstream primer 0.8 mu L, MYD88 probe 0.4 mu L, MYD88 wild blockPrimer 0.8 mu L, NAPDH upstream primer 0.3 mu L, NAPDH downstream primer 0.3 mu L, NADPH Probe 0.2 mu L, AceQ U + Probe Master Mix 12 mu L, ddH2O4. mu.L, template 5. mu.L.
Preferably, the conditions for PCR amplification are: 10min at 95 ℃; 95 ℃ for 30s, 62 ℃ for 1min, 35 cycles.
The invention has the beneficial effects that:
(1) the sensitivity is high: mutations as low as 0.12% could be detected.
(2) The operation is simple and convenient, and the template is added. The reagent contains dUTP/UNG enzyme anti-pollution system, and is closed-tube detection.
(3) The mutation rate of MYD88L265P can be quantitatively detected.
Drawings
FIG. 1: amplification curves for primer-probe ratios 1: 1.
FIG. 2: amplification curves for primer-probe ratios 1: 2.
FIG. 3: amplification curves for primer-probe ratios 1: 4.
FIG. 4: linear examination of mutant plasmid standards.
FIG. 5: linear examination of wild plasmid standards.
FIG. 6: repeat imprecision-29%.
FIG. 7: repeat imprecision-2%.
FIG. 8: and (4) detecting the range.
FIG. 9: the lowest detection limit.
FIG. 10: the samples are linear.
Detailed Description
It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.
As described in the background section, the incidence of the MYD88L265P mutation in Waldenstrom macro-globulinemia (WM) was more than 90% and is an important marker for diagnosis and differential diagnosis of WM. Therefore, the method has great significance for detecting the mutation MYD88L 265P.
Based on this, the invention aims to provide a kit for quantitatively detecting the mutation rate of MYD88L 265P.
The kit for quantitatively detecting the mutation rate of MYD88L265P provided by the invention comprises a standard substance for preparing a standard curve, a MYD88L265P wild type PCR system and a MYD88L265P mutant type PCR system.
The MYD88L265P wild type PCR system comprises a MYD88 wild upstream primer, a MYD88 downstream primer, a MYD88 probe, a MYD88 mutation blocking primer, an upstream primer and a downstream primer of an internal reference gene and a Taqman probe; the MYD88 mutant PCR system comprises a MYD88 mutant upstream primer, a MYD88 downstream primer, a MYD88 probe, a MYD88 wild closed primer, an upstream primer and a downstream primer of an internal reference gene and a Taqman probe.
According to the invention, NADPH is preferably used as an internal reference for monitoring the quality of a DNA template and judging whether amplification is smoothly carried out.
In order to make the technical solutions of the present application more clearly understood by those skilled in the art, the technical solutions of the present application will be described in detail below with reference to specific embodiments.
The test materials used in the examples of the present invention are all conventional in the art and commercially available. The experimental procedures, for which no detailed conditions are indicated, were carried out according to the usual experimental procedures or according to the instructions recommended by the supplier.
Example 1: amplification System optimization
Due to the technical characteristics of AS-PCR, the nonspecific amplification of the sample is stronger under high concentration, and a closed primer and a probe are introduced into the system for inhibiting the nonspecific amplification. MYD88L265P mutant negative samples were added for amplification using different probe and blocking primer ratios (1:1, 1:2, 1:4, molar ratio), and it was found that when the probe and blocking primer molar ratio was 1:2, non-specific amplification of mutant lines decreased and amplification of wild-type systems was not affected (fig. 1-fig. 3).
Example 2: construction of mutant, wild plasmid standards
Selecting amplified fragments and amplified fragments according to designed upstream and downstream primersAbout 100bp of the upstream and downstream of the segment are used for constructing a mutant plasmid and a wild plasmid respectively. The sequences of the mutant plasmid and the wild plasmid are respectively shown as SEQ ID NO.10 and SEQ ID NO. 11. Calculating copy number according to plasmid mass, and preparing mutant plasmid and wild plasmid into 5 × 106copies/. mu.l (S1), then treating S1 with process water at a rate of 1: 9 to obtain S2, S3 and S4 at 5X 10 concentrations5copies/μl、5×104copies/μl、5×103copies/mu l, and amplifying the standard substance by using an optimized system to verify the linearity, R2See fig. 4 and 5 for 1.
Example 3: kit composition and method of use
The kit comprises the following components:
table 1: kit Components
The sequences of each primer probe in MYD88L265P mutant premix and MYD88L265P wild premix were as follows:
table 2: primer probe sequence
Secondly, the use method comprises the following steps:
1. test sample
20 negative clinical samples and 20 positive clinical samples determined by the reference method.
2. Extraction of sample DNA
Human genome DNA is extracted by a whole blood nucleic acid extraction kit (Qiagen), the concentration and the purity of the nucleic acid (the concentration of the DNA is more than or equal to 10 ng/mu l, and the ratio of OD260/OD280 is between 1.8 and 2.0) are detected by a spectrophotometer, and then the DNA template for experiments is diluted to 75 ng/mu l. The method comprises the following specific steps:
(1) a sufficient amount of 1.5ml of the tube was taken and labeled, and 20. mu.l of the Protease (or the Protease K) solution was added to the bottom of the tube.
(2) After mixing the samples, 200. mu.l of the sample, e.g., less than 200. mu.l of the sample, was added to each tube and 200. mu.l of PBS was added to make up the sample.
(3) Add 200. mu.l of buffer AL and vortex for 15 s.
(4) The mixture is incubated at 56 ℃ for 10 minutes.
(5) Centrifuge briefly to spin off the tube walls and cover with solution.
(6) Add 200. mu.l ethanol (96-100%) to each tube, vortex for 15s, and centrifuge briefly.
(7) The liquid was carefully transferred to a QIAamp Mini spin column (placed in a 2ml collection tube). Centrifuge at 8,000rpm or more for 1min, place QIAamp Mini spin column into a clean 2ml collection tube, and discard the tube.
(8) Add 500. mu.l of the rinse AW1 (not wetting the edge of the tube), centrifuge at 8,000rpm for 1min, place the QIAamp Mini spin column into a clean 2ml collection tube, and discard the tube.
(9) Add 500. mu.l of the rinse AW2 (not wetting the edge of the tube), centrifuge at maximum speed (13,200rpm) for 3min, place the QIAamp Mini spin column into a clean 2ml collection tube, and discard the tube.
(10) The QIAamp Mini spin column was placed in a new tube (supplied by itself) and centrifuged at the highest speed (13,200rpm) for 1 min.
(11) The QIAamp Mini spin column was transferred to a new EP tube (1.5 ml), 50 to 200. mu.l of elution buffer AE or distilled water was suspended in the center of the adsorption membrane, and the membrane was left at room temperature (15 to 25 ℃) for 5 minutes and centrifuged at 8,000rpm (. about.13,400 Xg) for 1 minute.
(12) The DNA concentration and the OD260/OD280 ratio were measured using a Nanodrop instrument, and the template DNA was diluted with AE to a concentration of 75 ng/. mu.l.
PCR amplification
(1) And (3) taking the MYD88L265P mutant premix, the MYD88L265P wild premix, the mutant standard substance and the wild standard substance out of the kit, carrying out negative and positive quality control, melting at room temperature, mixing uniformly, and centrifuging at 2000rpm for 10 s.
(2) The required parts of reagents are as follows: n (number of samples) +2 (quality control) +4 (standard), 20. mu.l each of the mutant premix and the wild premix was dispensed according to Table 3.
Table 3: reagent split charging form
| The column is a sudden change premix | The extract is a wild premix | The column is a sudden change premix | The extract is a wild premix | |
| Mutant standard S1 | Wild | Sample | 1 | |
| Mutant standard S2 | Wild | Sample | 2 | |
| Mutant standard S3 | Wild | Sample | 3 | |
| Mutant standard S4 | Wild | Sample | 4 | |
| Negative quality control | Negative | Sample | 5 | |
| Positive quality control | Positive quality control | Sample 6 | Sample 6 | |
| Sample 7 | Sample 7 | |||
| |
|
(3) Sample adding: and adding the mixed standard substance, quality control substance and sample into the subpackaged reagents, and adding 5 mu l of sample into each PCR tube. The total volume of each reaction system was 25. mu.l, and the reaction system for mutant amplification and the reaction system for wild amplification were shown in Table 4 and Table 5, respectively. The tube cover is covered, centrifuged at 2000rpm for 10s, and moved to the amplification area for amplification. The instrument is recommended for ABI 7500 and amplification is performed using FAM and HEX channels, as shown in table 6.
Table 4: MYD88 mutant premix system
Table 5: MYD88 wild premix system
Table 6: PCR amplification procedure
4. Interpretation of results
(1) Determining baseline and threshold values:
the section with smaller fluctuation and more stable fluorescence curve is selected as the baseline, and can be adjusted according to the needs. The threshold value should be set at the inflection point of the amplification curve and cover the highest point of the non-amplification curve, and the negative control is not detected.
(2) And (3) judging the effectiveness:
negative quality control in FAM and HEX channels without amplification or Ct>34; positive quality control FAM channel CTThe value should be less than or equal to 34. At least one fluorescence signal is required to be arranged in a FAM or HEX channel of any detection hole of a sample to be detected; c of HEX signal of sample detection hole if FAM signal does not existTThe value is less than or equal to 30. If the above conditions are not satisfied, it is indicated that the DNA quality is not good or that the PCR reaction inhibitor is contained, and DNA is extracted again or sampling is required again.
(3) Negative or positive determination of the result
FAM Signal C at the lowest concentration of all wild and mutant reference products under FAM channelTThe value is less than or equal to 34, and all the wild-type detection holes of the samples are amplified. If the FAM signal of the sample mutant type detection hole is less than or equal to 34 under the condition of meeting the conditions, judging the sample mutant type detection hole to be positive; if the FAM signal is greater than 34, or "Undet" is displayed, it is judged to be negative.
(4) Quantification and calculation of mutation rates:
the mutant plasmid reference is used to determine the mutant copy number of the sample and the wild plasmid reference is used to determine the wild copy number of the sample.
Mutation rate ═ mutant copy number of samples/(mutant copy number of samples + wild copy number of samples) × 100%.
Example 4: methodology validation
1. Accuracy of
By adopting the kit, 20 health examination population samples which are subjected to mutation negative of digital PCR MYD88L265P and 20 MYD88L265P mutation positive patient samples are selected for detection, and the results are shown in Table 7, and the two methods are 100% consistent.
Table 7: correct comparison
2. Precision verification
(1) Repeatability imprecision: the mutant plasmid was mixed with the negative clinical specimen as shown in table 8, and the resulting samples with mutation rates of about 29% and 2% for MYD88L265P were tested in 20 replicates. Results as shown in tables 9 and 10 below, and fig. 6 and 7, MYD88L265P mutation rates were 29% and 2% for both samples of mutant and wild CTThe coefficient of variation (CV,%) of the values is 0.34 percent and 0.44 percent, 0.68 percent and 0.61 percent respectively, and the requirement of repeatability imprecision is met (CV is less than or equal to 5 percent).
Table 8: sample preparation method
Table 9: repeat imprecision-mutation Rate 29%
| Test number | Mutant C T | Wild T | Mutant copy number | Number of wild copies | Mutation Rate (%) | Interpretation results |
| H1 | 24.89 | 24.60 | 33180 | 78985 | 29.581 | Mutations |
| H2 | 24.98 | 24.70 | 31435 | 74294 | 29.731 | Mutations |
| H3 | 24.91 | 24.72 | 32891 | 73605 | 30.885 | Mutations |
| H4 | 24.88 | 24.69 | 33414 | 74630 | 30.926 | Mutations |
| H5 | 24.96 | 24.66 | 31863 | 75885 | 29.572 | Mutations |
| H6 | 24.96 | 24.62 | 31867 | 77822 | 29.052 | Mutations |
| H7 | 24.93 | 24.58 | 32428 | 79942 | 28.858 | Mutations |
| H8 | 24.88 | 24.51 | 33516 | 83309 | 28.689 | Mutations |
| H9 | 24.96 | 24.71 | 31858 | 73778 | 30.158 | Mutations |
| H10 | 25.00 | 24.68 | 31081 | 75292 | 29.219 | Mutations |
| H11 | 25.00 | 24.60 | 30929 | 78697 | 28.213 | Mutations |
| H12 | 24.95 | 24.70 | 32059 | 74135 | 30.189 | Mutations |
| H13 | 25.14 | 24.87 | 28331 | 67116 | 29.682 | Mutations |
| H14 | 25.08 | 24.92 | 29503 | 65156 | 31.168 | Mutations |
| H15 | 25.19 | 24.78 | 27466 | 71020 | 27.888 | Mutations |
| H16 | 25.02 | 24.84 | 30507 | 68335 | 30.864 | Mutations |
| H17 | 25.00 | 24.74 | 31077 | 72339 | 30.051 | Mutations |
| H18 | 25.09 | 24.82 | 29338 | 69266 | 29.753 | Mutations |
| H19 | 25.07 | 24.83 | 29592 | 68857 | 30.058 | Mutations |
| H20 | 25.01 | 24.80 | 30769 | 69948 | 30.550 | Mutations |
| Mean value of | 24.99 | 24.72 | / | / | / | / |
| SD | 0.08 | 0.11 | / | / | / | / |
| CV | 0.34％ | 0.44％ | / | / | / | / |
Table 10: repeat imprecision-mutation Rate 2%
(2) Intermediate imprecision: samples with mutation rates of approximately 29% and 2% as described above were used. The test was performed once a day, and 5 tests were repeated for each concentration for 4 consecutive days. Results as shown in tables 11 and 12 below, MYD88B L265P mutation rates were 29% and 2% of the mutant and wild C of the samples in the 4-day continuous testTThe CV value is far less than 5%, and meets the requirement of intermediate imprecision (CV is less than or equal to 5%). In addition, the mutation rates of both samples were very close to 29% and 2% of the original values, with only very small range fluctuations.
Table 11: intermediate imprecision degree-29%
Table 12: intermediate imprecision degree of-2%
2. Detection range and minimum detection limit
The mutant plasmids diluted in multiple proportion were mixed with MYD88L265P negative samples in proportion to obtain a series of samples L1-L8 with different mutation rates, and the preparation method is shown in Table 13. The detection is repeated for 3 times for each sample, the detection result is shown in table 14 and fig. 8, the mutation rate can be stably detected by 3 times for the L1-L8 samples, and the log value of the copy number is in an allowable range. The samples were prepared so that the final concentration of mutant copies of MYD88L265P in the samples was 75copies/μ L, the mutation rate was 0.12%, and the assay was repeated 20 times. As a result, as shown in FIG. 9 and Table 15, the detection was stable for 20 cycles.
Table 13: sample preparation method
Table 14: detection range
Table 15: minimum detection limit
| Detection number | Mutant C T | Wild T | Mutant copy number | Number of wild copies | Total number of | Mutation rate | |
| 1 | 32.67 | 24.67 | 115 | 86546 | 86661 | 0.133 | |
| 2 | 32.92 | 24.84 | 98 | 77996 | 78094 | 0.125 | |
| 3 | 33.21 | 24.92 | 81 | 74530 | 74611 | 0.108 | |
| 4 | 33.41 | 24.96 | 71 | 72541 | 72612 | 0.098 | |
| 5 | 33.04 | 24.98 | 90 | 71657 | 71747 | 0.126 | |
| 6 | 33.53 | 24.98 | 65 | 71765 | 71830 | 0.091 | |
| 7 | 32.61 | 24.87 | 120 | 76808 | 76928 | 0.156 | |
| 8 | 33.01 | 24.94 | 92 | 73482 | 73574 | 0.125 | |
| 9 | 33.01 | 24.77 | 92 | 81326 | 81418 | 0.113 | |
| 10 | 33.29 | 24.93 | 76 | 73939 | 74016 | 0.103 | |
| 11 | 33.14 | 24.88 | 84 | 76147 | 76231 | 0.111 | |
| 12 | 32.85 | 24.75 | 102 | 82347 | 82450 | 0.124 | |
| 13 | 32.79 | 24.71 | 107 | 84236 | 84343 | 0.126 | |
| 14 | 33.03 | 24.67 | 91 | 86242 | 86333 | 0.105 | |
| 15 | 33.19 | 24.79 | 82 | 80181 | 80263 | 0.102 | |
| 16 | 33.01 | 24.92 | 92 | 74396 | 74488 | 0.124 | |
| 17 | 32.72 | 24.80 | 112 | 79830 | 79942 | 0.140 | |
| 18 | 33.21 | 24.80 | 81 | 79869 | 79950 | 0.101 | |
| 19 | 33.19 | 24.76 | 82 | 81970 | 82052 | 0.100 | |
| 20 | 32.67 | 24.77 | 116 | 81243 | 81358 | 0.142 |
3. Linearity
Samples L1-L6 diluted at double ratios in the validation of detection range were used, and the assay was repeated 3 times per sample, as the average of log copy number and CTCalculating linear correlation coefficient by averaging values, requiring r2Not less than 0.980. As shown in Table 16 and FIG. 10, this verification r21.000, linearity meets the requirement.
Table 16: sample linearity
The above description is only a preferred embodiment of the present application and is not intended to limit the present application, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present application shall be included in the protection scope of the present application.
SEQUENCE LISTING
<110> Jinan-Ji-Min-Lai-Korea medical inspection center, Inc
<120> kit for detecting MYD88L265P mutation based on AS-PCR and application thereof
<130> 2020
<160> 11
<170> PatentIn version 3.5
<210> 1
<211> 24
<212> DNA
<213> Artificial sequence
<400> 1
<210> 2
<211> 24
<212> DNA
<213> Artificial sequence
<400> 2
ttgccttgta cttgatgggg attg 24
<210> 3
<211> 24
<212> DNA
<213> Artificial sequence
<400> 3
ttgccttgta cttgatgggg attg 24
<210> 4
<211> 24
<212> DNA
<213> Artificial sequence
<400> 4
<210> 5
<211> 21
<212> DNA
<213> Artificial sequence
<400> 5
gggttgaaga ctgggcttgt c 21
<210> 6
<211> 24
<212> DNA
<213> Artificial sequence
<400> 6
gcatcaggcc cttgccccat ggtg 24
<210> 7
<211> 20
<212> DNA
<213> Artificial sequence
<400> 7
cccactcctc cacctttgac 20
<210> 8
<211> 18
<212> DNA
<213> Artificial sequence
<400> 8
<210> 9
<211> 29
<212> DNA
<213> Artificial sequence
<400> 9
cattgccctc aacgaccact ttgtcaagc 29
<210> 10
<211> 360
<212> DNA
<213> mutant plasmid
<400> 10
tttgtgtgag tgaatgtgtg ccaggggtac ttagatgggg gatggctgtt gttaaccctg 60
gggttgaaga ctgggcttgt cccaccatgg ggcaagggcc tgatgccagc atggcacccc 120
ttggcttgca ggtgcccatc agaagcgacc gatccccatc aagtacaagg caatgaagaa 180
agagttcccc agcatcctga ggttcatcac tgtctgcgac tacaccaacc cctgcaccaa 240
atcttggttc tggactcgcc ttgccaaggc cttgtccctg ccctgaagac tgttctgagg 300
ccctgggtgt gtgtgtatct gtctgcctgt ccatgtactt ctgccctgcc tcctcctttc 360
<210> 11
<211> 360
<212> DNA
<213> wild plasmid
<400> 11
tttgtgtgag tgaatgtgtg ccaggggtac ttagatgggg gatggctgtt gttaaccctg 60
gggttgaaga ctgggcttgt cccaccatgg ggcaagggcc tgatgccagc atggcacccc 120
ttggcttgca ggtgcccatc agaagcgact gatccccatc aagtacaagg caatgaagaa 180
agagttcccc agcatcctga ggttcatcac tgtctgcgac tacaccaacc cctgcaccaa 240
atcttggttc tggactcgcc ttgccaaggc cttgtccctg ccctgaagac tgttctgagg 300
ccctgggtgt gtgtgtatct gtctgcctgt ccatgtactt ctgccctgcc tcctcctttc 360
Claims (10)
1. A primer-probe combination for detecting MYD88L265P mutations, comprising: a primer-probe combination A for detecting MYD88L265P mutant and a primer-probe combination B for detecting MYD88L265P wild type;
the primer-probe combination A comprises: a MYD88 wild blocking primer shown in SEQ ID NO.1, a MYD88 mutation upstream primer shown in SEQ ID NO.3, a MYD88 downstream primer shown in SEQ ID NO.5 and a MYD88 probe shown in SEQ ID NO. 6;
the primer-probe combination B comprises: MYD88 mutation blocking primer shown in SEQ ID NO.2, MYD88 wild upstream primer shown in SEQ ID NO.4, MYD88 downstream primer shown in SEQ ID NO.5 and MYD88 probe shown in SEQ ID NO. 6.
2. Use of the primer-probe combination of claim 1 in the preparation of a reagent and/or kit for detecting a mutation in MYD88L 265P.
3. A kit for detecting MYD88L265P mutation, wherein the kit comprises the primer-probe combination of claim 1.
4. The kit according to claim 3, further comprising: an NADPH upstream primer shown in SEQ ID NO.7, an NADPH downstream primer shown in SEQ ID NO.8 and an NADPH probe shown in SEQ ID NO. 9.
5. The kit according to claim 3, further comprising: mutant plasmid standards and wild plasmid standards; the nucleotide sequence of the mutant plasmid standard substance is shown as SEQ ID NO.10, and the nucleotide sequence of the wild plasmid standard substance is shown as SEQ ID NO. 11.
6. The kit according to any one of claims 3 to 5, wherein the primer-probe combination A, the NADPH upstream primer represented by SEQ ID NO.7, the NADPH downstream primer represented by SEQ ID NO.8, and the NADPH probe represented by SEQ ID NO.9 are previously mixed in one PCR reaction tube; the primer-probe combination B, the NADPH upstream primer shown in SEQ ID NO.7, the NADPH downstream primer shown in SEQ ID NO.8, and the NADPH probe shown in SEQ ID NO.9 were mixed in advance in another PCR reaction tube.
7. Use of the primer-probe combination of claim 1 or the kit of any one of claims 3-5 for predicting the efficacy of a drug for the treatment of macroglobulinemia fahrenheit.
8. A method of detecting a MYD88L265P mutation comprising the steps of:
respectively constructing a wild type amplification system and a mutant type amplification system by using DNA of a sample to be detected as a template and adopting the kit of any one of claims 3-5, and respectively carrying out PCR amplification by using the wild type amplification system and the mutant type amplification system; and quantifying the mutation copy number of the sample to be detected by using the mutation plasmid standard substance, quantifying the wild copy number of the sample to be detected by using the wild plasmid standard substance, and calculating to obtain the mutation rate of MYD88L 265P.
9. According to claim 8The method of (a), wherein the wild-type amplification system comprises: MYD88 wild upstream primer 0.8 mu L, MYD88 downstream primer 0.8 mu L, MYD88 Probe 0.4 mu L, MYD88 mutation blocking primer 0.8 mu L, NAPDH upstream primer 0.3 mu L, NAPDH downstream primer 0.3 mu L, NADPH Probe 0.2 mu L, AceQ U + Probe Master Mix 12 mu L, ddH2O4.0 μ L, template 5 μ L;
the mutant amplification system comprises: MYD88 mutation upstream primer 0.8 mu L, MYD88 downstream primer 0.8 mu L, MYD88 Probe 0.4 mu L, MYD88 wild blocking primer 0.8 mu L, NAPDH upstream primer 0.3 mu L, NAPDH downstream primer 0.3 mu L, NADPH Probe 0.2 mu L, AceQ U + Probe Master Mix 12 mu L, ddH2O4.0. mu.L, template 5. mu.L.
10. The method of claim 8, wherein the PCR amplification conditions are: 10min at 95 ℃; 95 ℃ for 30s, 62 ℃ for 1min, 35 cycles.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110189489.1A CN112831556A (en) | 2021-02-19 | 2021-02-19 | Kit for detecting MYD88L265P mutation based on AS-PCR and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110189489.1A CN112831556A (en) | 2021-02-19 | 2021-02-19 | Kit for detecting MYD88L265P mutation based on AS-PCR and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN112831556A true CN112831556A (en) | 2021-05-25 |
Family
ID=75933867
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110189489.1A Pending CN112831556A (en) | 2021-02-19 | 2021-02-19 | Kit for detecting MYD88L265P mutation based on AS-PCR and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112831556A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2023282179A1 (en) * | 2021-07-06 | 2023-01-12 | 東洋鋼鈑株式会社 | Microarray kit, and target detection method using microarray |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110229910A (en) * | 2019-07-08 | 2019-09-13 | 福建省肿瘤医院（福建省肿瘤研究所、福建省癌症防治中心） | MYD88 gene L265P mutation detection kit and detection method |
| CN110438223A (en) * | 2018-05-03 | 2019-11-12 | 同济大学苏州研究院 | Detect primer, probe and its kit and detection method of Kras point mutation |
| CN110951860A (en) * | 2019-12-23 | 2020-04-03 | 济南金域医学检验中心有限公司 | Method for detecting JAK2V617F mutation rate and special primer and probe thereof |
| CN110982898A (en) * | 2019-10-14 | 2020-04-10 | 苏州艾可瑞斯生物科技有限公司 | MyD88 gene L265P mutation detection kit based on ARMS-PCR and detection method |
| CN111593126A (en) * | 2020-06-22 | 2020-08-28 | 杭州千麦医学检验所有限公司 | Primer and probe for detecting MYD88 gene L265P mutation and high-sensitivity detection method |
-
2021
- 2021-02-19 CN CN202110189489.1A patent/CN112831556A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110438223A (en) * | 2018-05-03 | 2019-11-12 | 同济大学苏州研究院 | Detect primer, probe and its kit and detection method of Kras point mutation |
| CN110229910A (en) * | 2019-07-08 | 2019-09-13 | 福建省肿瘤医院（福建省肿瘤研究所、福建省癌症防治中心） | MYD88 gene L265P mutation detection kit and detection method |
| CN110982898A (en) * | 2019-10-14 | 2020-04-10 | 苏州艾可瑞斯生物科技有限公司 | MyD88 gene L265P mutation detection kit based on ARMS-PCR and detection method |
| CN110951860A (en) * | 2019-12-23 | 2020-04-03 | 济南金域医学检验中心有限公司 | Method for detecting JAK2V617F mutation rate and special primer and probe thereof |
| CN111593126A (en) * | 2020-06-22 | 2020-08-28 | 杭州千麦医学检验所有限公司 | Primer and probe for detecting MYD88 gene L265P mutation and high-sensitivity detection method |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2023282179A1 (en) * | 2021-07-06 | 2023-01-12 | 東洋鋼鈑株式会社 | Microarray kit, and target detection method using microarray |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Setty et al. | A pyrosequencing-based assay for the rapid detection of IDH1 mutations in clinical samples | |
| CN114085903B (en) | Primer pair probe combination product for detecting mitochondria 3243A & gtG mutation, kit and detection method thereof | |
| CN103421909A (en) | Genetic diagnosis reagent for Citrin deficiency disease and application of genetic diagnosis reagent | |
| CN108456726A (en) | Spinal muscular atrophy genetic test probe, primer and kit | |
| CN110951860A (en) | Method for detecting JAK2V617F mutation rate and special primer and probe thereof | |
| CN108277281A (en) | A kind of kit and its detection method of detection people's B-raf genes V600E mutation | |
| CN117070607A (en) | Fluorescent quantitative PCR kit for detecting human spinal muscular atrophy SMN1 and SMN2 gene copy numbers | |
| CN106498028B (en) | Diagnostic method and kit for T790M mutation of EGFR | |
| CN112831556A (en) | Kit for detecting MYD88L265P mutation based on AS-PCR and application thereof | |
| CN103993089B (en) | A kind of multiple fluorescence quantitative PCR detection kit and detection method thereof of vancomycin-resistant enterococcus | |
| CN108130362A (en) | Kit and application for EGFR genetic mutation detection | |
| CN111500687A (en) | Breast cancer 21 gene detection kit | |
| CN111304329A (en) | Kit for detecting mutation of BRAF gene V600E site | |
| CN116024303A (en) | EML4-ALK fusion gene quantitative genome RNA standard substance and preparation method thereof | |
| CN110144386A (en) | For detecting the primer, probe and kit of POLE gene mutation | |
| CN115851935A (en) | Primer probe set and kit for MET gene exon14 jump mutation detection | |
| CN109306374A (en) | For detecting primer, detection method and the kit of mankind PIK3CA gene E545K mutation | |
| CN111793675B (en) | Nucleic acid sequence for EGFR gene 18 exon G719X mutation detection, kit and application thereof | |
| CN111621557B (en) | Kit for detecting osteoporosis susceptibility gene by cocktail and using method thereof | |
| CN112143815B (en) | Nucleic acid composition, kit and detection method for detecting fusion mutation of human FGFR2 gene | |
| CN106811537A (en) | One kind detection epidermal growth factor receptor gene T790M low frequencies mutant primer and its application | |
| CN110295232B (en) | microRNA biomarkers for colorectal cancer | |
| Bai et al. | Establishment and validation of the detection of TERT promoter mutations by human gliomas U251 cell lines | |
| CN111705132A (en) | Primer probe set, kit and method for detecting liver cancer prognosis marker TP53R249S by ddPCR (double-stranded polymerase chain reaction) | |
| CN113502332B (en) | Primer, probe and kit for detecting FLT3 gene mutation |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |