CN111593126A - Primer and probe for detecting MYD88 gene L265P mutation and high-sensitivity detection method - Google Patents
Primer and probe for detecting MYD88 gene L265P mutation and high-sensitivity detection method Download PDFInfo
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Abstract
The invention discloses a primer and a probe for detecting MYD88 gene L265P mutation and a high-sensitivity detection method, wherein the detection process comprises the following steps: step one, extracting DNA in a human whole blood sample; step two, performing fluorescence PCR detection to obtain a fluorescence result graph: specifically amplifying wild DNA by using MYD 88L 265P F, MYD 88L 265P WR primers and MYD 88L 265P Probe; specifically amplifying mutant DNA by using MYD 88L 265P F and MYD 88L 265P MR primers and MYD 88L 265P Probe Probe; thirdly, judging a mutation result by using a fluorescence result graph; the invention utilizes AS-PCR fluorescence method, and utilizes the primer and probe obtained by screening, thus improving the specificity and sensitivity of detection.
Description
Technical Field
The invention relates to the field of gene mutation detection, in particular to a primer and a probe for detecting MYD88 gene L265P mutation and a high-sensitivity detection method.
Background
The myelinoid differentiation primary response 88 (MYD 88) gene is located on the short arm of chromosome 3 and has a total of 5 exons. The mutation of MYD 88L 265P gene is a T → C point mutation of MYD88 gene at position 38182640 of exon 5, which results in the replacement of leucine at amino acid 265 of TIR region of MYD88 protein by proline, and is the most dominant mutation of MYD88 gene.
MYD88 gene mutations were found in approximately 90% LPL/WM (lymphoplasmacytic lymphoma/Waldenstrom macroglobulinemia). While SMZL (splenic marginal zone lymphoma), CLL plasma cell differentiation and IgM secretory MM have rare or even no MYD88 gene mutation. Therefore, the gene mutation can be used for the differential diagnosis of LPL/WM.
In addition, MYD88 gene mutation is also found in IgM-sectioning MGUS (monoclonal immunoglobulin blood disease with unknown IgM type significance), and the mutation rate is 60% -80%. Meanwhile, the MYD88 gene mutation is related to prognosis through research. MYD88 gene mutation predicts slow disease progression and longer survival in LPL/WM patients compared to wild type; and is a risk factor in IgM-secreting MGUS patients, with higher serum IgM levels, more bone marrow invasion; the risk of developing WM increases.
Currently, the following methods are common for MYD 88L 265P mutation detection:
PCR-Sanger sequencing method
Currently, the most common method for detecting gene mutation is adopted for multiple studies to detect MYD88 mutation. However, most LPL/WM patients have abnormal bone marrow and peripheral blood cell ratio lower than 30% in initial diagnosis, while the sensitivity of the conventional Sanger sequencing method is 15% -20%, and the conventional PCR-Sanger method may have false negative sequencing results.
AS-PCR electrophoresis method
The detection method is also commonly used in gene detection similar to single base mutation at present. Compared with the traditional PCR-Sanger sequencing method, the AS-PCR electrophoresis method has the advantages of higher sensitivity and shorter detection period. However, the AS-PCR electrophoresis method, which adopts electrophoresis detection, is easy to cause result misjudgment in a sample with low mutation content.
The market needs a rapid, high-sensitivity and high-specificity detection method for detecting MYD 88L 256P mutation, and the invention solves the problem.
Disclosure of Invention
In order to solve the defects of the prior art, the invention aims to provide a primer and a probe for detecting MYD88 gene L265P mutation and a high-sensitivity detection method.
In order to achieve the above object, the present invention adopts the following technical solutions:
a primer and a probe for detecting MYD88 gene L265P mutation,
the primer comprises:
MYD88 L265P F:GGATGGCTGTTGTTAACC；
MYD88 L265P WR1:GCCTTGTACTTGATGGGGATgA；
MYD88 L265P MR1:CCTTGTACTTGATGGGGATgG；
the probe includes:
MYD88 L265P Probe:5’FAM-CCCTTGGCTTGCAGGTGCCCATCAGA-3’TAMRAM。
a high-sensitivity detection method for detecting MYD88 gene L265P mutation comprises the following steps:
step one, extracting DNA in a human whole blood sample;
step two, performing fluorescence PCR detection to obtain a fluorescence result graph:
specifically amplifying wild DNA by using MYD 88L 265P F and MYD 88L 265P WR1 primers and MYD 88L 265P Probe probes;
specifically amplifying mutant DNA by using MYD 88L 265P F and MYD 88L 265P MR1 primers and MYD 88L 265P Probe Probe;
the primer comprises:
MYD88 L265P F:GGATGGCTGTTGTTAACC；
MYD88 L265P WR1:GCCTTGTACTTGATGGGGATgA；
MYD88 L265P MR1:CCTTGTACTTGATGGGGATgG；
the probe includes:
MYD88 L265P Probe:5’FAM-CCCTTGGCTTGCAGGTGCCCATCAGA-3’TAMRAM；
step three, judging whether the result is mutated according to the amplification curve graphs of the MYD88 wild amplification system and the mutation amplification system;
the interpretation of the results is based on the following principles:
note: MM stands for the result of the mutant amplification system, MW for the result of the wild amplification system.
The high-sensitivity detection method for detecting the mutation of the MYD88 gene L265P,
the configured PCR amplification system comprises: wild type system, mutant system;
the wild-type system comprises:
primers MYD 88L 265P F and MYD 88L 265P WR1 are each 0.4-1ul,
MYD 88L 265P Probe (10. mu.M) 0.2-0.6ul,
PCR Master Mix 12.5ul，
4.9-10.5ul of deionized water,
1-5ul of DNA template;
the mutant system comprises:
primers MYD 88L 265P F and MYD 88L 265P MR1 are 0.4-1ul each,
MYD 88L 265P Probe (10. mu.M) 0.2-0.6ul,
Realtime PCR Master Mix 12.5ul，
4.9-10.5ul of deionized water,
the DNA template is 1-5 ul.
The high-sensitivity detection method for detecting the mutation of the MYD88 gene L265P,
the wild-type system comprises:
primers MYD 88L 265P F and MYD 88L 265P WR1 each 0.8ul,
MYD 88L 265P Probe (10. mu.M) 0.4ul,
PCR Master Mix 12.5ul，
8.5ul of deionized water is added,
2ul of DNA template;
the high-sensitivity detection method for detecting the mutation of the MYD88 gene L265P,
the mutant system comprises:
primers MYD 88L 265P F and MYD 88L 265P MR1 each 0.8ul,
MYD 88L 265P Probe (10. mu.M) 0.4ul,
Realtime PCR Master Mix 12.5ul，
8.5ul of deionized water is added,
the DNA template was 2 ul.
The high-sensitivity detection method for detecting the mutation of the MYD88 gene L265P,
step two, performing fluorescence PCR detection to obtain a fluorescence result graph:
the conditions for PCR amplification were:
the invention has the advantages that:
according to the invention, an AS-PCR fluorescence method is utilized, and the primers and the probes obtained by screening are utilized in a matched manner, so that the detection specificity is improved, 10 cases of healthy human blood samples are detected, and the positive result is not amplified; the sensitivity of detection is improved, and positive bone marrow samples with abnormal cell content as low as 2.6 percent can still be detected by flow cytometry.
The detection process speed is only 2-2.5 hours to complete the detection.
Drawings
FIG. 1 is a graph showing the result of MYD 88L 265P locus sequencing on a blood sample A to be detected by using a sequencing method;
FIG. 2 is a graph showing the result of MYD 88L 265P locus detection in a blood sample A to be detected by the method of the present invention;
FIG. 3 is a graph showing the result of MYD 88L 265P locus sequencing on blood sample B to be detected by using a sequencing method;
FIG. 4 is a graph showing the result of MYD 88L 265P locus detection on a blood sample A to be detected by using a sequencing method;
FIG. 5 is a graph showing the fluorescence results of MYD88 gene L265P mutation detection in a blood sample A to be detected by using the AS-PCR fluorescence method of the present invention;
FIG. 6 is a graph showing the result of MYD 88L 265P locus sequencing on blood sample B to be detected by using a sequencing method;
FIG. 7 is a graph showing the fluorescence results of MYD88 gene L265P mutation detection in a blood sample B to be detected using the AS-PCR fluorescence method of the present invention;
FIG. 8 is a schematic diagram of the amplification results of the specificity verification experiment of the present invention.
Detailed Description
The invention is described in detail below with reference to the figures and the embodiments.
A primer and a probe for detecting MYD88 gene L265P mutation,
the primer comprises:
MYD88 L265P F:GGATGGCTGTTGTTAACC；
MYD88 L265P WR1:GCCTTGTACTTGATGGGGATgA；
MYD88 L265P MR1:CCTTGTACTTGATGGGGATgG；
the probe includes:
MYD88 L265P Probe:5’FAM-CCCTTGGCTTGCAGGTGCCCATCAGA-3’TAMRAM。
the screening process for such primer probes is as follows:
according to the MYD 88L 265P mutation position, the following sets of specific primers are designed in combination with the influence of base substitution on the specificity of the primers:
selecting sequencing positive samples AS positive samples, selecting healthy controls AS negative samples, and performing AS-PCR amplification, wherein the detection result is shown in figure 1;
as can be seen from fig. 1: in the first scheme, the positive sample, the negative sample mutation amplification system and the wild amplification system can both see strips, and in the third scheme, the positive sample, the negative sample mutation amplification system and the wild amplification system both fail to amplify; in the second scheme, the positive sample mutation amplification system and the wild amplification system can both see strips, the negative sample wild amplification system can both see strips, and the mutation amplification system has no strips.
According to the experimental result, the scheme II is reserved, and a fluorescent probe is further designed:
MYD88 L265P Probe:5’FAM-CCCTTGGCTTGCAGGTGCCCATCAGA-3’TAMRAM。
and (3) verification experiment:
firstly, extracting DNA in a human whole blood sample for the following detection experiment;
the human blood DNA extraction process is as follows: blood Genomic Mini Kit Blood genome column type miniprep Kit (kang century Biotechnology Ltd.) was used. Adding a 200 mu 1 whole blood sample to be detected into an I.5ml centrifuge tube, adding a 20 mu 1 proteinase K solution, and uniformly mixing by oscillation; add 200. mu.1 buffer GL, shake until thorough mixing, stand lOmin at 56 ℃ and mix 3 times by inversion. Adding 200 μ 1 anhydrous ethanol, mixing, adding the solution into adsorption column (Spin Columns DM) filled in collecting tube, centrifuging at 12000rpm for 1min, and removing waste liquid; adding 500 mu 1 buffer solution GW1 into the adsorption column, centrifuging at 12000rpm for 1min, and discarding the waste liquid; adding 600 mu 1 rinsing liquid GW2 into the adsorption column, centrifuging at 12000rpm for 1min, and discarding the waste liquid; and repeating the steps once. Then the adsorption column is put back into the collection tube, and is subjected to air centrifugation at 12000rpm for 2 min; and transferring the adsorption column into a clean centrifugal tube, suspending and dropwise adding 50-100 mu I GE to the middle part of the adsorption membrane, standing at room temperature for 2-5min, centrifuging at 12000rpm for 2min, collecting the solution into the centrifugal tube to obtain the sample DNA, and using the sample DNA in the following experiments.
Experiment I, the mutation of MYD88 gene L265P is detected by using the AS-PCR fluorescence method, and the specific process is AS follows:
the method comprises the following steps: and (3) fluorescent PCR detection: the PCR amplification system was prepared with the following reagents and reagents amounts,
wild type system:
wherein each of primers MYD 88L 265P F and MYD 88L 265P WR is 0.8ul, and MYD 88L 265P Probe (10 μ M) is 0.4 ul; realtime PCR Master Mix 12.5ul (Toyobo (Shanghai) science and technology Co., Ltd.); adding 8.5ul of deionized water; finally, 2ul of DNA template is added.
Mutant system:
wherein each of the primers MYD 88L 265P F and MYD 88L 265P MR is 0.8ul, and the MYD 88L 265P Probe (10 uM) is 0.4 ul; realtime PCR Master Mix 12.5ul (Toyobo (Shanghai) science and technology Co., Ltd.); adding 8.5ul of deionized water; finally, 2ul of DNA template is added.
The primer comprises:
MYD88 L265P F:GGATGGCTGTTGTTAACC；
MYD88 L265P WR1:GCCTTGTACTTGATGGGGATgA；
MYD88 L265P MR1:CCTTGTACTTGATGGGGATgG；
the probe includes:
MYD88 L265P Probe:5’FAM-CCCTTGGCTTGCAGGTGCCCATCAGA-3’TAMRAM。
the PCR amplification conditions were:
and step two, judging whether the result is mutated according to the amplification curve graphs of the MYD88 wild amplification system and the mutation amplification system.
In general, amplification curves were seen for the MYD 88L 265P mutant samples, both wild amplification system and mutant amplification system, as shown in fig. 2:
MYD 88L 265P wild-type samples, only the wild-type amplification system seen the amplification curve, as shown in fig. 3:
the interpretation of the results is based on the following principles:
note: MM stands for the result of the mutant amplification system, MW for the result of the wild amplification system.
The fluorescence result of the blood sample A to be tested is shown in FIG. 5.
The fluorescence result of the blood sample B to be examined is shown in FIG. 7.
And (4) analyzing results: the samples A and B to be detected can both have amplification curves in a wild amplification system and a mutation amplification system, and the CT values are both less than 36, so that the samples A and B to be detected can be judged to be MYD 88L 265P mutation samples.
Experiment two, specificity verification experiment;
10 samples of the healthy normal physical examination are taken, and DNA is extracted according to the steps.
The human blood DNA extraction process is as follows: blood Genomic Mini Kit Blood genome column type miniprep Kit (kang century Biotechnology Ltd.) was used. Adding a 200 mu 1 whole blood sample to be detected into an I.5ml centrifuge tube, adding a 20 mu 1 proteinase K solution, and uniformly mixing by oscillation; add 200. mu.1 buffer GL, shake until thorough mixing, stand lOmin at 56 ℃ and mix 3 times by inversion. Adding 200 μ 1 anhydrous ethanol, mixing, adding the solution into adsorption column (Spin Columns DM) filled in collecting tube, centrifuging at 12000rpm for 1min, and removing waste liquid; adding 500 mu 1 buffer solution GW1 into the adsorption column, centrifuging at 12000rpm for 1min, and discarding the waste liquid; adding 600 mu 1 rinsing liquid GW2 into the adsorption column, centrifuging at 12000rpm for 1min, and discarding the waste liquid; and repeating the steps once. Then the adsorption column is put back into the collection tube, and is subjected to air centrifugation at 12000rpm for 2 min; and transferring the adsorption column into a clean centrifugal tube, suspending and dropwise adding 50-100 mu I GE to the middle part of the adsorption membrane, standing at room temperature for 2-5min, centrifuging at 12000rpm for 2min, collecting the solution into the centrifugal tube to obtain the sample DNA, and using the sample DNA in the following experiments.
And (3) fluorescent PCR detection: the PCR amplification system is configured according to the following reagents and reagent amounts, wherein 12 parts of wild type and mutant type systems respectively (10 detection samples + positive control (positive sample for sequencing detection) + blank control)
Wild type system:
wherein each of primers MYD 88L 265P F and MYD 88L 265P WR is 0.8ul, and MYD 88L 265P Probe (10 μ M) is 0.4 ul; realtime PCR Master Mix 12.5ul (Toyobo (Shanghai) science and technology Co., Ltd.); adding 8.5ul of deionized water; finally, 2ul of DNA template is added.
Mutant system:
wherein each of the primers MYD 88L 265P F and MYD 88L 265P MR is 0.8ul, and the MYD 88L 265P Probe (10 uM) is 0.4 ul; realtime PCR Master Mix 12.5ul (Toyobo (Shanghai) science and technology Co., Ltd.); adding 8.5ul of deionized water; finally, 2ul of DNA template is added.
The primer comprises:
MYD88 L265P F:GGATGGCTGTTGTTAACC；
MYD88 L265P WR1:GCCTTGTACTTGATGGGGATgA；
MYD88 L265P MR1:CCTTGTACTTGATGGGGATgG；
the probe includes:
MYD88 L265P Probe:5’FAM-CCCTTGGCTTGCAGGTGCCCATCAGA-3’TAMRAM。
the PCR amplification conditions were:
and (4) analyzing results: the amplification result is shown in FIG. 8, 10 samples of healthy human are detected, positive amplification is not generated, and the method improves the specificity of detection.
Comparison experiment one: sequencing the MYD 88L 265P locus of the blood sample A to be detected by using a sequencing method, wherein the sequencing process is as follows:
firstly, PCR amplification: the PCR amplification system was prepared with the following reagents and reagents in amounts of 0.4ul each of primers MYD 88L 265P F and MYD 88L 265P R (10. mu.M), 2 XKOD Buffer 10ul, KOD enzyme (1U/ul)0.4ul, dNTP (2mM)2ul (Toyobo (Shanghai) Biotech Co., Ltd.); adding deionized water 5.8 ul; finally, 1ul of DNA template is added.
The PCR primers were as follows:
| MYD88 L265P F | TGTGAGTGAATGTGTGCCAG |
| MYD88 L265P R | AAGGCGAGTCCAGAACCAAG |
the amplification procedure was as follows:
secondly, PCR product purification: the enzyme purification was configured as follows: CIP (NEB Co.) 0.1ul, Exo I (NEB Co.) 0.5ul, deionized water 1.4 ul; finally 2ul were added. The purification reaction procedure is as follows: 45min at 37 ℃ and 5min at 98 ℃.
Thirdly, sequencing reaction: 5ul per reaction system:
terminator v3.1 enzyme plus 0.4ul,
Terminator v3.1 Buffer with 0.6ul primer, 3.2P primer fixed with 1ul primer, template with 2ul primer after purification, then adding corresponding 1ul primer into water, and then adding 1ul BigDye mixed solution at different places of the tube wall; covering a tube cover, centrifuging at a low speed for a short time, uniformly mixing by using a vortex device, and centrifuging at a low speed for a short time again; according to the following reaction conditions, pre-denaturation is carried out for 4min at 95 ℃; denaturation at 95 ℃ for 15s, annealing at 50 ℃ for 20s, extension at 68 ℃ for 2min, 25 cycles. And (5) loading the PCR kit to a PCR instrument for sequencing reaction.
Thirdly, purifying sequencing products: carrying out short-time centrifugation on the amplified sequencing reaction product; adding 2ul of 125mM EDTA into each tube, covering a cover, performing vortex mixing, performing short-time centrifugation, adding 15ul of absolute ethyl alcohol, and performing vortex mixing; selecting program 2(4000rpm, centrifuging for 30min) by using a plate centrifuge, then carrying out inversion instantaneous centrifugation at 300rpm, and removing supernatant; adding 50ul of pre-cooled 75% ethanol at-20 deg.C, vortex mixing, selecting program 4 (centrifuging at 4000rpm and 4 deg.C for 15 min); performing inversion at 300rpm for instantaneous centrifugation, removing supernatant, standing at room temperature for 15min, and volatilizing ethanol; after the ethanol is completely volatilized, 10ul of Hi-Di are added into each tubeTMFully vortexing and oscillating for 1min, standing for 15min after short-time centrifugation to completely dissolve sequencing products; Hi-Di is addedTMThe tubes are arranged correspondingly according to the primers, and the sample numbers on the tubes are correspondingly recorded into a sample table according to the arranged sequence. Preparing a sequencer for use; each tube with the sample weaved will put Hi-Di according to the position in the sampleTMTransfer of lysates into corresponding positions in 96-well plates; placing the 96-well plate on a PCR amplification instrument, performing pre-denaturation at 95 ℃ for 5min, and quickly transferring the plate to an ice box to cool the plate for 5min at 4 ℃; and (4) carrying out machine sequencing on a sequencer.
FIG. 4 shows the result chart of the blood sample A to be tested. Flow cytometry indicated a bone marrow B lymphocyte ratio of 2.60%. As can be seen from comparison with the result of sample A of experiment I, the method of the present invention has improved detection sensitivity, and positive bone marrow samples with abnormal cell content as low as 2.6% can still be detected by flow cytometry.
Comparative experiment two: sequencing a MYD 88L 265P locus of a blood sample B to be detected by using a sequencing method;
the specific process is shown in comparative experiment I.
FIG. 6 shows the result chart of the blood sample B to be tested: flow cytometry indicated a bone marrow B lymphocyte fraction of 10.56%.
And (4) analyzing results: an AS-PCR Taqman fluorescence method is used for respectively designing primers aiming at an L265P mutant type and a wild type of a MYD88 gene in DNA. The basic principle is to design two specific primers, the last base at the 3' end of the primer is the same as the wild type or mutant base, and the other primer is a universal forward primer. When PCR reaction is carried out, because Taq DNA polymerase lacks 3' -5' exonuclease activity, if the 3' end of a primer forms mismatch, chain extension reaction is blocked, and a specific amplification product cannot be detected in a certain amplification cycle; on the contrary, the 3' end pairing can detect the amplification product; meanwhile, a Taqman probe is added, so that the specificity of a detection result is further improved. Compared with a Sanger sequencing method, the method has the advantages that the detection cost is saved, the detection period is greatly shortened, the detection efficiency is improved, and the detection sensitivity is also greatly improved.
The foregoing illustrates and describes the principles, general features, and advantages of the present invention. It should be understood by those skilled in the art that the above embodiments do not limit the present invention in any way, and all technical solutions obtained by using equivalent alternatives or equivalent variations fall within the scope of the present invention.
Claims (6)
1. A primer and a probe for detecting MYD88 gene L265P mutation are characterized in that,
the primer comprises:
MYD88 L265P F:GGATGGCTGTTGTTAACC；
MYD88 L265P WR1:GCCTTGTACTTGATGGGGATgA；
MYD88 L265P MR1:CCTTGTACTTGATGGGGATgG；
the probe includes:
MYD88 L265P Probe:5’FAM-CCCTTGGCTTGCAGGTGCCCATCAGA-3’TAMRAM。
2.a high-sensitivity detection method for detecting MYD88 gene L265P mutation is characterized by comprising the following steps:
step one, extracting DNA in a human whole blood sample;
step two, performing fluorescence PCR detection to obtain a fluorescence result graph:
specifically amplifying wild DNA by using MYD 88L 265P F and MYD 88L 265P WR1 primers and MYD 88L 265P Probe probes;
specifically amplifying mutant DNA by using MYD 88L 265P F and MYD 88L 265P MR1 primers and MYD 88L 265P Probe Probe;
the primer comprises:
MYD88 L265P F:GGATGGCTGTTGTTAACC；
MYD88 L265P WR1:GCCTTGTACTTGATGGGGATgA；
MYD88 L265P MR1:CCTTGTACTTGATGGGGATgG；
the probe includes:
MYD88 L265P Probe:5’FAM-CCCTTGGCTTGCAGGTGCCCATCAGA-3’TAMRAM；
step three, judging whether the result is mutated according to the amplification curve graphs of the MYD88 wild amplification system and the mutation amplification system;
the interpretation of the results is based on the following principles:
note: MM stands for the result of the mutant amplification system, MW for the result of the wild amplification system.
3. The high sensitivity detection method for detecting MYD88 gene L265P mutation of claim 2,
the configured PCR amplification system comprises: wild type system, mutant system;
the wild-type system comprises:
primers MYD 88L 265P F and MYD 88L 265P WR1 are each 0.4-1ul,
MYD 88L 265P Probe (10. mu.M) 0.2-0.6ul,
PCR Master Mix 12.5ul，
4.9-10.5ul of deionized water,
1-5ul of DNA template;
the mutant system comprises:
primers MYD 88L 265P F and MYD 88L 265P MR1 are 0.4-1ul each,
MYD 88L 265P Probe (10. mu.M) 0.2-0.6ul,
Realtime PCR Master Mix 12.5ul，
4.9-10.5ul of deionized water,
the DNA template is 1-5 ul.
4. The high sensitivity detection method of MYD88 gene L265P mutation of claim 3, wherein the mutation is detected by the mutation detection method,
the wild-type system comprises:
primers MYD 88L 265P F and MYD 88L 265P WR1 each 0.8ul,
MYD 88L 265P Probe (10. mu.M) 0.4ul,
PCR Master Mix 12.5ul，
8.5ul of deionized water is added,
the DNA template was 2 ul.
5. The high sensitivity detection method of MYD88 gene L265P mutation of claim 3, wherein the mutation is detected by the mutation detection method,
the mutant system comprises:
primers MYD 88L 265P F and MYD 88L 265P MR1 each 0.8ul,
MYD 88L 265P Probe (10. mu.M) 0.4ul,
Realtime PCR Master Mix 12.5ul，
8.5ul of deionized water is added,
the DNA template was 2 ul.
6. The high sensitivity detection method for detecting MYD88 gene L265P mutation of claim 2,
step two, performing fluorescence PCR detection to obtain a fluorescence result graph:
the conditions for PCR amplification were:
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