[go: up one dir, main page]

CN118421769B - Multiplex PCR primer group for detecting quality of samples used for second-generation sequencing and application thereof - Google Patents

Multiplex PCR primer group for detecting quality of samples used for second-generation sequencing and application thereof Download PDF

Info

Publication number
CN118421769B
CN118421769B CN202410874608.0A CN202410874608A CN118421769B CN 118421769 B CN118421769 B CN 118421769B CN 202410874608 A CN202410874608 A CN 202410874608A CN 118421769 B CN118421769 B CN 118421769B
Authority
CN
China
Prior art keywords
primer
detecting
seq
generation sequencing
multiplex pcr
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.)
Active
Application number
CN202410874608.0A
Other languages
Chinese (zh)
Other versions
CN118421769A (en
Inventor
杨成镐
陈光辉
孙建龙
丁波
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Shanghai Fudison Life Sciences Co ltd
Original Assignee
Shanghai Fudison Life Sciences Co ltd
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Shanghai Fudison Life Sciences Co ltd filed Critical Shanghai Fudison Life Sciences Co ltd
Priority to CN202410874608.0A priority Critical patent/CN118421769B/en
Publication of CN118421769A publication Critical patent/CN118421769A/en
Application granted granted Critical
Publication of CN118421769B publication Critical patent/CN118421769B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

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
    • C12Q1/6869—Methods for sequencing
    • 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
    • C12Q1/686—Polymerase chain reaction [PCR]

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Health & Medical Sciences (AREA)
  • Biophysics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Immunology (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Analytical Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

The invention relates to the field of gene detection, in particular to a multiplex PCR primer group for detecting the quality of samples used for second-generation sequencing and application thereof. The multiplex PCR primer set provided by the invention comprises primers for detecting various genes as follows: MTOR, ALK, PIK3CA, FAT1, APC, ROS1, EGFR, MYC, NOTCH1, RET, WT1, KRAS, AKT1 or TP53. The multiplex PCR primer group provided by the invention can be used for evaluating the quality of the whole genome amplification yield of 1-10 human cells by amplifying specific regions of specific genes, is beneficial to judging whether a sample is worth performing downstream NGS sequencing, and avoids subsequent sequencing failure or poor quality, thereby causing larger time and money loss.

Description

Multiplex PCR primer group for detecting quality of samples used for second-generation sequencing and application thereof
Technical Field
The invention relates to the field of gene detection, in particular to a multiplex PCR primer group for detecting the quality of samples used for second-generation sequencing and application thereof.
Background
Currently existing whole genome amplification (Whole genome amplification, WGA) techniques are mainly multiplex strand displacement (Multiple displacement amplification, MDA). First, a random six-base primer anneals to the template DNA at multiple sites, and then the psi 29 DNA polymerase simultaneously initiates replication at multiple sites of the template DNA, which synthesizes DNA along the DNA template while replacing the complementary strand of the template. The displaced complementary strand becomes a new template for amplification, and thus a large amount of high molecular weight DNA can be finally obtained.
For single cells with particularly small template initiation amounts (6 pg DNA), it is challenging to perform WGA on them. Firstly, the MDA method cannot amplify the whole genome completely and uniformly, resulting in low amplification efficiency of partial regions; second, since the amount of template is small, the success of amplification will depend on the quality of single cells, and if the cells are not fresh or damaged, the failure of amplification is easily caused.
Therefore, it is necessary to evaluate the quality of the whole genome amplification products of cells prior to downstream NGS analysis, avoiding subsequent sequencing failures or poor data quality, resulting in greater time and monetary losses.
Disclosure of Invention
In view of the above-described drawbacks of the prior art, an object of the present invention is to provide a multiplex PCR primer set for detecting the quality of samples for second generation sequencing and use thereof, for solving the problems in the prior art.
To achieve the above and other related objects, the present invention provides a multiplex PCR primer set for detecting the quality of a sample for second generation sequencing, the multiplex PCR primer set comprising primers for detecting a plurality of genes as follows: MTOR, ALK, PIK3CA, FAT1, APC, ROS1, EGFR, MYC, NOTCH1, RET, WT1, KRAS, AKT1 or TP53.
Preferably, the multiplex PCR primer group comprises any of a plurality of primer pairs with nucleotide sequences shown as SEQ ID No.1-36, and the primer pair for detecting KRAS comprises a primer with nucleotide sequences shown as SEQ ID No. 1-2; the primer pair for detecting ALK comprises a primer with a nucleotide sequence shown as SEQ ID No. 3-4; the primer pair for detecting PIK3CA comprises a primer with a nucleotide sequence shown as SEQ ID No. 5-6; the primer pair for detecting FAT1 comprises a primer with a nucleotide sequence shown as SEQ ID No. 7-8; the primer pair for detecting the APC comprises a primer with a nucleotide sequence shown as SEQ ID No. 9-10; the primer pair for detecting the ROS1 comprises a primer with a nucleotide sequence shown as SEQ ID No. 11-12; the primer pair for detecting EGFR comprises a primer with a nucleotide sequence shown as SEQ ID No. 13-14; the primer pair for detecting MYC comprises a primer with a nucleotide sequence shown as SEQ ID No. 15-16; the primer pair for detecting NOTCH1 comprises a primer with a nucleotide sequence shown as SEQ ID No. 17-18; the primer pair for detecting RET comprises a primer with a nucleotide sequence shown as SEQ ID No. 19-20; the primer pair for detecting the WT1 comprises primers with nucleotide sequences shown as SEQ ID No. 21-22; the primer pair for detecting KRAS comprises primers with nucleotide sequences shown as SEQ ID No.23-24, SEQ ID No.29-30 or SEQ ID No. 35-36; the primer pair for detecting AKT1 comprises primers with nucleotide sequences shown as SEQ ID No.25-26 or SEQ ID No. 31-32; the primer pair for detecting TP53 comprises primers with nucleotide sequences shown as SEQ ID Nos. 27-28 or SEQ ID Nos. 33-34.
The invention also provides application of the multiplex PCR primer group in preparing a sample quality product for detecting second-generation sequencing.
The invention also provides a detection kit for detecting the quality of samples used for second-generation sequencing, which comprises the multiplex PCR primer group.
The invention also provides a method for detecting the quality of a sample used for second generation sequencing, which comprises the following steps:
1) Amplifying a sample for second generation sequencing to be detected by using the multiplex PCR primer group;
2) Counting the number of the amplified products in the step 1), and judging whether the sample used for the second-generation sequencing to be detected meets the requirement of the second-generation sequencing on the sample according to the number of the amplified products.
As described above, the multiplex PCR primer set for detecting the quality of samples used for second-generation sequencing and the use thereof have the following beneficial effects:
The multiplex PCR primer group provided by the invention can be used for evaluating the quality of the whole genome amplification yield of 1-10 human cells by amplifying specific regions of specific genes, is beneficial to judging whether a sample is worth performing downstream NGS sequencing, and avoids subsequent sequencing failure or poor quality, thereby causing larger time and money loss.
Drawings
FIG. 1 is a graph showing the amplification results of the initial multiplex PCR primer set of the present invention. Wherein, the DNA template used in lane 1 is human leukocyte DNA extracted by commercial kit; lane 2 is 100-1500 bp DNA Marker; lane 3 is 50-700 bp DNA Marker.
FIG. 2 is a graph showing the amplification results of the adjusted multiplex PCR primer set according to the present invention. Wherein lane 1 is 50-700 bp DNA Marker; the DNA template used in lane 2 was human leukocyte DNA extracted by commercial kits; lane 3 is 100-1500 bp DNA Marker.
FIG. 3 is a graph showing the amplification results of the final multiplex PCR primer set of the present invention. Wherein lane 1 is 100-1500 bp DNA Marker; lane 2 uses DNA template that is the product of WGA from 10 HepG2 cell line cells; lane 3 uses DNA template that is the product of 10 NCI-H1975 cell lines after WGA; lane 4 uses DNA templates that are human leukocyte DNA extracted with commercial kits; lane 5 is 50-700 bp DNA Marker.
FIG. 4 shows the results of verifying that there is a correlation between high quality levels and downstream NGS sequencing data quality in the present invention. Wherein lane 1 is 50-700 bp DNA Marker; lane 2 uses DNA template that is the product of WGA from 10 healthy human leukocytes; lane 3 uses DNA template that is product #3 of 1 healthy human leukocyte after WGA; lane 4 uses DNA templates that are human leukocyte DNA extracted with commercial kits; lane 5 uses DNA template that is product #2 of 1 healthy human leukocyte after WGA; lane 6 uses DNA template that is the WGA-derived product #1 of 1 healthy human leukocyte; lane 7 is 100-1500 bp DNA Marker.
FIG. 5 shows the results of the present invention verifying that there is a correlation between low quality levels and downstream NGS sequencing data quality. Wherein lane 1 is 100-1500 bp DNA Marker; the DNA template used in lane 2 is the WGA-derived product of 10 CTCs (Circulating Tumor Cells ) whose genome was destroyed after chemoradiotherapy; the DNA template used in lane 3 was human leukocyte DNA extracted by commercial kits.
Detailed Description
The invention provides a multiplex PCR primer group for detecting the quality of samples used for second-generation sequencing, which comprises primers for detecting the following various genes: MTOR, ALK, PIK3CA, FAT1, APC, ROS1, EGFR, MYC, NOTCH1, RET, WT1, KRAS, AKT1 or TP53.
In some embodiments, the multiplex PCR primer sets amplify a plurality of genes as follows: MTOR, ALK, PIK 3A, FAT1, APC, ROS1, EGFR, MYC, NOTCH1, RET, WT1, KRAS, AKT1 or TP53 amplified fragments are all of non-uniform length.
In some embodiments, the amplified fragment of the primer for detecting MTOR is 80-120bp in length. Specifically, the length of the fragment amplified by the primer for detecting MTOR is 80-90, 90-95, 95-100, 100-105, 105-110 or 110-120bp. Preferably, the amplified fragment of the primer for detecting MTOR has a length of 90-110bp.
In some embodiments, the amplified fragment of the primer for detecting ALK is 130-170bp in length. Specifically, the length of the fragment amplified by the primer for detecting ALK is 130-140, 140-145, 145-150, 150-155, 155-160 or 160-170bp. Preferably, the amplified fragment of the primer for detecting MTOR has a length of 140-160bp.
In some embodiments, the amplified fragment of the primer for detecting PIK3CA is 180-220bp in length. Specifically, the length of the fragment amplified by the primer for detecting PIK3CA is 180-190, 190-195, 195-200, 200-205, 205-210 or 210-220bp. Preferably, the length of the fragment amplified by the primer for detecting PIK3CA is 190-210bp.
In some embodiments, the length of the fragment amplified by the primer for detecting FAT1 is 230-270bp. Specifically, the length of the fragment amplified by the primer for detecting FAT1 is 230-240, 240-245, 245-250, 250-255, 255-260 or 260-270bp. Preferably, the length of the fragment amplified by the primer for detecting FAT1 is 240-260bp.
In some embodiments, the fragment amplified by the primer for detecting APC is 280-320bp in length. Specifically, the length of the fragment amplified by the primer for detecting APC is 280-290, 290-295, 295-300, 300-305, 305-310 or 310-320bp. Preferably, the length of the fragment amplified by the primer for detecting APC is 290-310bp.
In some embodiments, the primer for detecting ROS1 amplifies a fragment of 370-430bp in length. Specifically, the length of the fragment amplified by the primer for detecting ROS1 is 370-380, 380-390, 390-400, 400-410, 410-420 or 420-430bp. Preferably, the primer for detecting ROS1 will amplify a fragment of 390-410bp in length.
In some embodiments, the amplified fragment of the primer for detecting EGFR is 470-530bp in length. Specifically, the length of the fragment amplified by the EGFR-detecting primer is 470-480, 480-490, 490-500, 500-510, 510-520 or 520-530bp. Preferably, the length of the fragment amplified by the EGFR-detecting primer is 490-510bp.
In some embodiments, the length of the amplified fragment of the primer for detecting MYC is 570-630bp. Specifically, the length of the fragment amplified by the primer for detecting MYC is 570-580, 580-590, 590-600, 600-610, 610-620 or 620-630bp. Preferably, the length of the amplified fragment of the primer for detecting MYC is 590-610bp.
In some embodiments, the amplified fragment of a primer for detecting NOTCH1 is 670-730bp in length. Specifically, the length of the fragment amplified by the primer for detecting NOTCH1 is 670-680, 680-690, 690-700, 700-710, 710-720 or 720-730bp. Preferably, the primers for detecting NOTCH1 will amplify fragments between 690 and 710bp in length.
In some embodiments, the length of the amplified fragment of the RET-detecting primer is 770-830bp. Specifically, the length of the fragment amplified by the RET-detecting primer is 770-780, 780-790, 790-800, 800-810, 810-820 or 820-830bp. Preferably, the length of the fragment amplified by the RET-detecting primer is 790-810bp.
In some embodiments, the length of the fragment amplified by the primer for detecting WT1 is 870-930bp. Specifically, the length of the fragment amplified by the primer for detecting WT1 is 870-880, 880-890, 890-900, 900-910, 910-920 or 920-930bp. Preferably, the length of the fragment amplified by the primer for detecting WT1 is 890-910bp.
In some embodiments, the length of the amplified fragment of the primer for detection of KRAS is 970-1030bp. Specifically, the length of fragments amplified by the KRAS detection primers is 970-980, 980-990, 990-1000, 1000-1010, 1010-1020 or 1020-1030bp. Preferably, the length of the fragment amplified by the primer for detecting KRAS is 990-1010bp.
In some embodiments, the length of the fragment amplified by the primer for detecting AKT1 is 1200-1300bp. Specifically, the length of the amplified fragment of the primer for detecting AKT1 is 1200-1220, 1220-1240, 1240-1250, 1250-1260, 1260-1280 or 1280-1300bp. Preferably, the length of the fragment amplified by the primer for detecting AKT1 is 1240-1260bp.
In some embodiments, the amplified fragment of the primer for detecting TP53 is 1450-1550bp in length. Specifically, the length of the amplified fragment of the primer for detecting TP53 is 1450-1470, 1470-1490, 1490-1500, 1500-1510, 1510-1530 or 1530-1550bp. Preferably, the length of the fragment amplified by the primer for detecting TP53 is 1490-1510bp.
In some embodiments, the multiplex PCR primer set comprises any of the pairs of primers having the nucleotide sequences set forth in SEQ ID Nos. 1-36. Preferably, the multiplex PCR primer set comprises primer pairs having nucleotide sequences as shown in SEQ ID Nos. 1-28. Wherein, the primer pair for detecting KRAS comprises a primer with a nucleotide sequence shown as SEQ ID No. 1-2; the primer pair for detecting ALK comprises a primer with a nucleotide sequence shown as SEQ ID No. 3-4; the primer pair for detecting PIK3CA comprises a primer with a nucleotide sequence shown as SEQ ID No. 5-6; the primer pair for detecting FAT1 comprises a primer with a nucleotide sequence shown as SEQ ID No. 7-8; the primer pair for detecting the APC comprises a primer with a nucleotide sequence shown as SEQ ID No. 9-10; the primer pair for detecting the ROS1 comprises a primer with a nucleotide sequence shown as SEQ ID No. 11-12; the primer pair for detecting EGFR comprises a primer with a nucleotide sequence shown as SEQ ID No. 13-14; the primer pair for detecting MYC comprises a primer with a nucleotide sequence shown as SEQ ID No. 15-16; the primer pair for detecting NOTCH1 comprises a primer with a nucleotide sequence shown as SEQ ID No. 17-18; the primer pair for detecting RET comprises a primer with a nucleotide sequence shown as SEQ ID No. 19-20; the primer pair for detecting the WT1 comprises primers with nucleotide sequences shown as SEQ ID No. 21-22; the primer pair for detecting KRAS comprises primers with nucleotide sequences shown as SEQ ID No.23-24, SEQ ID No.29-30 or SEQ ID No. 35-36; the primer pair for detecting AKT1 comprises primers with nucleotide sequences shown as SEQ ID No.25-26 or SEQ ID No. 31-32; the primer pair for detecting TP53 comprises primers with nucleotide sequences shown as SEQ ID Nos. 27-28 or SEQ ID Nos. 33-34.
In some embodiments, the multiplex PCR primer set can be used to amplify DNA fragments formed after amplification of 1-10 whole genomes of a cell genome.
The invention also provides application of the multiplex PCR primer group in preparing a sample quality product for detecting second-generation sequencing.
The invention also provides a detection kit for detecting the quality of samples used for second-generation sequencing, which comprises the multiplex PCR primer group.
In some embodiments, the detection kit further comprises PCR reaction reagents. Specifically, the PCR reaction reagent comprises one or more of PCR buffer, DNA polymerase, magnesium ion, calcium ion, dNTPs, surfactant or preservative.
In some embodiments, the PCR buffer may be a phosphate buffer. The phosphate buffer is used to provide stable enzymatic reaction conditions. The surfactant is used for solubilization, and the dispersing solute promotes enzymatic reaction. The preservative is used to control the growth of microorganisms in the PCR reaction reagents.
The phosphate in the phosphate buffer solution is selected from potassium phosphate or sodium phosphate. The potassium phosphate salt and the sodium phosphate salt are selected from any one or more of potassium phosphate, monopotassium phosphate, dipotassium phosphate, sodium dihydrogen phosphate and disodium hydrogen phosphate.
The surfactant is a substance which contains hydrophilic groups and hydrophobic groups at the same time so as to obviously reduce the surface tension of the target solution. The surfactant is selected from any one or more of stearic acid, sodium dodecyl benzene sulfonate, quaternary ammonium compound, lecithin, amino acid type, betaine type, alkyl glucoside, fatty glyceride, fatty sorbitan or polysorbate.
The preservative may be self-produced or use commercial agents. Preferably, the preservative is any one or more of the commercial ProClin series of preservatives ProClin, 150, 200, 300, or 5000. More preferably, the preservative is ProClin300,300.
The invention also provides a method for detecting the quality of a sample used for second generation sequencing, which comprises the following steps:
1) Amplifying a sample to be tested for second-generation sequencing by using the multiplex PCR primer group;
2) Counting the number of the amplified products in the step 1), and judging whether the sample used for the second-generation sequencing to be detected meets the requirement of the second-generation sequencing on the sample according to the number of the amplified products.
In some embodiments, the amplification in step 1) is as follows: 90-105 ℃ 1-5 min, 20-40 s at 90-100 ℃, 20-40 s at 50-70 ℃ and 0.5-2 min at 65-75 ℃ are taken as one cycle, 30-40 cycles are carried out in total, and finally, 1-10min at 65-75 ℃.
In some embodiments, the sample used for second-generation sequencing to be tested in step 2) does not meet the requirements of second-generation sequencing on the sample when the number of amplified products is below 8, and meets the requirements of second-generation sequencing on the sample when the number of amplified products is above 9.
In some embodiments, the number of amplification products may be the number of bands generated by gel electrophoresis or the number of fragments of interest obtained by sequencing.
Other advantages and effects of the present invention will become apparent to those skilled in the art from the following disclosure, which describes the embodiments of the present invention with reference to specific examples. The invention may be practiced or carried out in other embodiments that depart from the specific details, and the details of the present description may be modified or varied from the spirit and scope of the present invention.
Before the embodiments of the invention are explained in further detail, it is to be understood that the invention is not limited in its scope to the particular embodiments described below; it is also to be understood that the terminology used in the examples of the invention is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the invention; in the description and claims of the invention, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.
Where numerical ranges are provided in the examples, it is understood that unless otherwise stated herein, both endpoints of each numerical range and any number between the two endpoints are significant both in the numerical range. 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 invention belongs. In addition to the specific methods, devices, materials used in the embodiments, any methods, devices, and materials of the prior art similar or equivalent to those described in the embodiments of the present invention may be used to practice the present invention according to the knowledge of one skilled in the art and the description of the present invention.
Example 1 screening of primers in multiplex PCR set
Primers as in Table 1 were synthesized, and multiplex PCR and agarose gel electrophoresis were performed according to the following procedure. The template is human leukocyte Total DNA extracted by a commercial kit.
TABLE 1
Primer name Sequence(s)
chr1-MTOR-F SEQ ID No.1:CTGCCTGAGGACTTTCCGAC
chr1-MTOR-R SEQ ID No.2:CATCCCTCAGTCTTGGTGCC
chr2-ALK-F SEQ ID No.3:CTGACTAGACTGGTAGAAAGGC
chr2-ALK-R SEQ ID No.4:CTTTGGCAATTAATCATGCCTGTG
chr3-PIK3CA-F SEQ ID No.5:AAAGGAAGCTCATTCCTCACC
chr3-PIK3CA-R SEQ ID No.6:TCAACTCACACTGTATGACATTTAC
chr4-FAT1-F SEQ ID No.7:AAACTGTCCGGCGTAATCACT
chr4-FAT1-R SEQ ID No.8:CAGTCCCACTATGTTCTGCTCG
chr5-APC-F SEQ ID No.9:GGGCCCGGTATTCATTCCTC
chr5-APC-R SEQ ID No.10:ACCCCAAGAGGCGATCATGT
chr6-ROS1-F SEQ ID No.11:GTGCCTCGTCACAATCATGC
chr6-ROS1-R SEQ ID No.12:AGGGCTTGATGCATGGCTTA
chr7-EGFR-F SEQ ID No.13:AAAGGCCATGCTCCCACAAT
chr7-EGFR-R SEQ ID No.14:AGTGGGTTGCACAGTGTCTC
chr8-MYC-F SEQ ID No.15:GGTAGGCGCGCGTAGTTAAT
chr8-MYC-R SEQ ID No.16:GGCAAAGTTTCGTGGATGCG
chr9-NOTCH1-F SEQ ID No.17:ATCCTCCTTGATCTGGGTGC
chr9-NOTCH1-R SEQ ID No.18:GTCCACATCGTACTGGCACA
chr10-RET-F SEQ ID No.19:GGAAATGAGGTGCTCGCTCT
chr10-RET-R SEQ ID No.20:TCATGCCATCGGTGTACCTG
chr11-WT1-F SEQ ID No.21:CTCCGCAGCGCTAAAATGTC
chr11-WT1-R SEQ ID No.22:AGGGCGGTTTCGATTTCTCC
chr12-KRAS-F SEQ ID No.29:ACCCAGCTAATGGTGTTCGG
chr12-KRAS-R SEQ ID No.30:CACTGGATTAAGAAGCAATGCCC
chr14-AKT1-F SEQ ID No.31:CCAGCTGATGAAGACGGAGC
chr14-AKT1-R SEQ ID No.32:ATGGAAAACCGGCCTAGGTG
chr17-TP53-F SEQ ID No.33:TGCTCAAGACTGGCGCTAAA
chr17-TP53-R SEQ ID No.34:CTGCGGAATGAAACAACCGC
1) The primer set 10 xWGA QC Mix was prepared as in Table 2, and the concentration of each primer was 100. Mu.M.
TABLE 2
Primer name Volume (mu L)
chr1-MTOR-F 3
chr1-MTOR-R 3
chr2-ALK-F 2.8
chr2-ALK-R 2.8
chr3-PIK3CA-F 2.5
chr3-PIK3CA-R 2.5
chr4-FAT1-F 2.2
chr4-FAT1-R 2.2
chr5-APC-F 1.9
chr5-APC-R 1.9
chr6-ROS1-F 1.6
chr6-ROS1-R 1.6
chr7-EGFR-F 1.4
chr7-EGFR-R 1.4
chr8-MYC-F 1.3
chr8-MYC-R 1.3
chr9-NOTCH1-F 1.2
chr9-NOTCH1-R 1.2
chr10-RET-F 1.1
chr10-RET-R 1.1
chr11-WT1-F 1
chr11-WT1-R 1
chr12-KRAS-F 0.9
chr12-KRAS-R 0.9
chr14-AKT1-F 0.8
chr14-AKT1-R 0.8
chr17-TP53-F 0.7
chr17-TP53-R 0.7
10 mM Tris-HCl pH 8.0 55.2
Totals to 100
2) The PCR system was configured as in table 3, positive control referring to fresh human Total DNA extracted by any commercial kit.
TABLE 3 Table 3
Reagent(s) Volume (mu L)
2× KAPA HiFi HotStart ReadyMix 10
10× WGA QC Mix 2
WGA product/positive control ~ 150 ng
PCR Grade Water Supplement to 8
3) Run PCR according to Table 4 procedure
TABLE 4 Table 4
Temperature (temperature) Cycle number Time of
95℃ 1 180 s
98℃ 35 20 s
62.5℃ 35 15 s
72℃ 35 30 s
72℃ 1 90 s
4℃ Maintenance of
4) Mu.L of multiplex PCR product was taken for each sample, and the gel was visualized or photographed under UV light on a 2% agarose gel pre-stained with GelRed at 120V electrophoresis 50 min.
As shown in FIG. 1, the band amplification efficiency was lower at 1000 and 1250 bp, and the corresponding primer sequences were redesigned as shown in Table 5.
TABLE 5
Primer name Sequence(s)
chr12-KRAS-F SEQ ID No.35:TTACCCAGCTAATGGTGTTCG
chr12-KRAS-R SEQ ID No.36:CTGGATTAAGAAGCAATGCCCTC
chr14-AKT1-F SEQ ID No.25:GCCTCTTCCATGAGAGGGTG
chr14-AKT1-R SEQ ID No.26:GGGGTGGCTTAGGTTGACTT
The above experimental steps were repeated to perform multiplex PCR and agarose gel electrophoresis, the results are shown in FIG. 2, the band amplification efficiency at 1000 and 1500 bp was low, and the corresponding primer sequences were redesigned as shown in Table 6.
TABLE 6
Primer name Sequence(s)
chr12-KRAS-F SEQ ID No.23:TACCCAGCTAATGGTGTTCG
chr12-KRAS-R SEQ ID No.24:ACTGGATTAAGAAGCAATGCCCT
chr17-TP53-F SEQ ID No.27:CCGGGACGTGAAAGGTTAGA
chr17-TP53-R SEQ ID No.28:CCAGGAAGGACAAAGGTCCG
10 NCI-H1975 cells, 10 WGA products of HepG2 cells, and human leukocyte Total DNA extracted by commercial kit were taken and subjected to multiplex PCR and agarose gel electrophoresis according to the above-described experimental procedures.
As shown in FIG. 3, all 14 amplicon bands were visible in the extracted leukocyte DNA, i.e., low sample size (samples formed from about 10 cell genomes) could also be amplified, indicating that the sensitivity of the primer set was satisfactory. Furthermore, all samples, except the expected band and a small amount of primer dimer <100 bp, had no apparent non-specific band generation and primer dimer length did not affect the observation of specific bands, i.e., no band generation, indicating that the specificity of the primer set was satisfactory.
Example 2 correlation verification of quality class and downstream second generation sequencing (NGS) sequencing data quality
Blood from a volunteer was drawn, three 1 white blood cells were picked, one 10 white blood cells were WGA, and white blood cell Total DNA was extracted as a positive control using a commercial kit. Five DNA products obtained were subjected to multiplex PCR and agarose gel electrophoresis according to the experimental procedure in example 1.
The results are shown in fig. 4, and the results of the evaluation of the respective essential quantities are as follows based on the results of fig. 4:
Sample name Deletion strip (bp) Number of bands successfully amplified Quality grade
1 WBC #1 100、150、700、900、1250 9 C
1 WBC #2 200 13 A
1 WBC #3 100、700 12 B
10 WBC 700 13 A
WBC extraction Without any means for 14 A
The 5 samples were subjected to whole-exome sequencing according to the standard procedure of sequencing manufacturer Illumina, and the partial sequencing quality control indexes are as follows (target region in the table refers to whole-exome region):
Sample name 1 WBC #1 1 WBC #2 1 WBC #3 10 WBC WBC extraction
Sequencing depth >10 target region ratio (%) 60.821 94.704 89.06 96.072 99.523
Average sequencing depth of target region 113.0793 191.9956 93.3589 161.771 256.0308
SNP quantity 50231 84566 78304 93002 99029
From the above results, the higher the quality level of the sample, the better the overall sequencing quality control index.
In order to demonstrate that the quality control primer set can also evaluate the amplification products of the whole genome, which are poorly amplified, blood from a volunteer with osteosarcoma, who had undergone multiple rounds of chemoradiotherapy, is withdrawn, and the genome of the cells in the blood has been destroyed. 10 CTC cells in blood were picked for WGA, while leukocyte Total DNA was extracted as a positive control using a commercial kit. The obtained DNA products were subjected to multiplex PCR and agarose gel electrophoresis according to the technical scheme, and the results are shown in FIG. 5 and Table 7.
TABLE 7
Sample name Deletion strip (bp) Number of bands successfully amplified Quality grade
CTC 100、150、200、250、300、400、500、700、900 5 F
The samples were subjected to whole-exome sequencing according to the standard procedure of sequencing manufacturer Illumina, and the partial sequencing quality control indexes are as follows (target region in the table refers to whole-exome region):
Sample name CTC
Sequencing depth >10 target region ratio (%) 0.15%
Average sequencing depth of target region 0.2694
SNP quantity 821
From the index of sequencing quality control, the CTC sample amplification is failed, and only a small portion of the exon regions are successfully amplified, so that the quality control primer set can evaluate the whole genome amplification products with poor amplification.
In summary, the quality class correlates with downstream NGS sequencing data quality. Therefore, samples can be classified as follows according to the number of bands successfully amplified, and samples with a class of C or more can be subjected to subsequent sequencing experiments.
The rating criteria in this example are shown in table 8.
TABLE 8
Number of strips Grade
13 - 14 A
11 - 12 B
9 - 10 C
8. Or below F
Explanation:
Class a: the product has good amplification quality, can be subjected to downstream analysis, and can be expected to obtain more ideal sequencing data.
B level: the product has better amplification quality, can be subjected to downstream analysis, and is expected to obtain sequencing data slightly worse than class A.
C level: the amplification quality of the product is generally such that risk downstream analysis can be performed, with poor sequencing data expected to be obtained.
Grade F: the amplification quality of the product was poor and downstream analysis was not suggested.
The above examples are provided to illustrate the disclosed embodiments of the invention and are not to be construed as limiting the invention. Further, various modifications of the methods set forth herein, as well as variations of the methods of the invention, will be apparent to those skilled in the art without departing from the scope and spirit of the invention. While the invention has been specifically described in connection with various specific preferred embodiments thereof, it should be understood that the invention should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in the art are intended to be within the scope of the present invention.

Claims (7)

1. A multiplex PCR primer set for detecting the quality of a sample of circulating tumor cells for use in second generation sequencing, said multiplex PCR primer set comprising primers for detecting a plurality of genes: MTOR, ALK, PIK3CA, FAT1, APC, ROS1, EGFR, MYC, NOTCH1, RET, WT1, KRAS, AKT1, or TP53;
The multiplex PCR primer set amplifies a plurality of genes as follows: MTOR, ALK, PIK 3A, FAT1, APC, ROS1, EGFR, MYC, NOTCH1, RET, WT1, KRAS, AKT1 or TP53 amplified fragments are all of non-uniform length;
The multiplex PCR primer group comprises primer pairs with nucleotide sequences shown as SEQ ID No. 1-28;
The multiplex PCR primer set comprises a plurality of pairs of primers as follows: the primer pair for detecting KRAS comprises a primer with a nucleotide sequence shown as SEQ ID No. 1-2; the primer pair for detecting ALK comprises a primer with a nucleotide sequence shown as SEQ ID No. 3-4; the primer pair for detecting PIK3CA comprises a primer with a nucleotide sequence shown as SEQ ID No. 5-6; the primer pair for detecting FAT1 comprises a primer with a nucleotide sequence shown as SEQ ID No. 7-8; the primer pair for detecting the APC comprises a primer with a nucleotide sequence shown as SEQ ID No. 9-10; the primer pair for detecting the ROS1 comprises a primer with a nucleotide sequence shown as SEQ ID No. 11-12; the primer pair for detecting EGFR comprises a primer with a nucleotide sequence shown as SEQ ID No. 13-14; the primer pair for detecting MYC comprises a primer with a nucleotide sequence shown as SEQ ID No. 15-16; the primer pair for detecting NOTCH1 comprises a primer with a nucleotide sequence shown as SEQ ID No. 17-18; the primer pair for detecting RET comprises a primer with a nucleotide sequence shown as SEQ ID No. 19-20; the primer pair for detecting the WT1 comprises primers with nucleotide sequences shown as SEQ ID No. 21-22; the primer pair for detecting KRAS comprises a primer with a nucleotide sequence shown as SEQ ID No. 23-24; the primer pair for detecting AKT1 comprises a primer with a nucleotide sequence shown as SEQ ID No. 25-26; the primer pair for detecting TP53 comprises primers with nucleotide sequences shown as SEQ ID No. 27-28.
2. Use of the multiplex PCR primer set as defined in claim 1 for the preparation of a sample quality product for detecting second generation sequencing.
3. A test kit for detecting the quality of a sample for use in second generation sequencing, said test kit comprising the multiplex PCR primer set according to claim 1.
4. The test kit of claim 3, wherein the test kit further comprises PCR reaction reagents.
5. The test kit of claim 4, wherein the PCR reaction reagents comprise one or more of PCR buffers, DNA polymerase, magnesium ions, calcium ions, dNTPs, surfactants, or preservatives.
6. A method of detecting the quality of a sample for use in second generation sequencing, said method comprising the steps of:
1) Amplifying a sample for second generation sequencing to be tested using the multiplex PCR primer set of claim 1;
2) Counting the number of the amplified products in the step 1), and judging whether the sample used for the second-generation sequencing to be detected meets the requirement of the second-generation sequencing on the sample according to the number of the amplified products.
7. The method according to claim 6, wherein the sample used for the second-generation sequencing to be tested does not satisfy the requirement of the second-generation sequencing on the sample when the number of the amplified products in the step 2) is less than 8, and the sample used for the second-generation sequencing to be tested satisfies the requirement of the second-generation sequencing on the sample when the number of the amplified products is more than 9.
CN202410874608.0A 2024-07-02 2024-07-02 Multiplex PCR primer group for detecting quality of samples used for second-generation sequencing and application thereof Active CN118421769B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202410874608.0A CN118421769B (en) 2024-07-02 2024-07-02 Multiplex PCR primer group for detecting quality of samples used for second-generation sequencing and application thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202410874608.0A CN118421769B (en) 2024-07-02 2024-07-02 Multiplex PCR primer group for detecting quality of samples used for second-generation sequencing and application thereof

Publications (2)

Publication Number Publication Date
CN118421769A CN118421769A (en) 2024-08-02
CN118421769B true CN118421769B (en) 2024-10-01

Family

ID=92323593

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202410874608.0A Active CN118421769B (en) 2024-07-02 2024-07-02 Multiplex PCR primer group for detecting quality of samples used for second-generation sequencing and application thereof

Country Status (1)

Country Link
CN (1) CN118421769B (en)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN118726591B (en) * 2024-09-03 2024-11-05 上海复迪生生命科学有限公司 A multiplex PCR primer set for detecting gastrointestinal cancer-related gene mutations and its use

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104762405A (en) * 2015-04-22 2015-07-08 北京嘉宝仁和医疗科技有限公司 Method and kit for quality appraisal for amplification products after single cell genome amplification
CN113584138A (en) * 2021-09-07 2021-11-02 广西壮族自治区妇幼保健院 Quality control method for MDA single cell whole genome amplification product
CN116769879A (en) * 2023-07-07 2023-09-19 中信湘雅生殖与遗传专科医院有限公司 Nucleic acid products, kits and methods for quality control of MDA amplification products
CN116926066A (en) * 2022-04-01 2023-10-24 上海明悦医疗科技有限公司 Primer combination product, kit for quality detection and genome quality detection method

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102533960B (en) * 2010-12-31 2014-04-30 深圳华大基因科技有限公司 Single-cell genome analysis method and kit
CN108315416A (en) * 2018-03-02 2018-07-24 中国科学院合肥物质科学研究院 Primer, kit and the method for lung cancer gene mutation site are determined based on high throughput sequencing technologies
CN117625750A (en) * 2022-08-19 2024-03-01 上海明悦医疗科技有限公司 Whole genome amplification primer compositions, kits and methods
CN116219036A (en) * 2023-02-23 2023-06-06 华南农业大学 Primer group, kit and method for quality control of cynomolgus monkey whole genome amplification
CN117844933B (en) * 2024-03-07 2024-06-11 上海复迪生生命科学有限公司 Multiplex PCR primer group for detecting lung tumor related gene variation and application thereof

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104762405A (en) * 2015-04-22 2015-07-08 北京嘉宝仁和医疗科技有限公司 Method and kit for quality appraisal for amplification products after single cell genome amplification
CN113584138A (en) * 2021-09-07 2021-11-02 广西壮族自治区妇幼保健院 Quality control method for MDA single cell whole genome amplification product
CN116926066A (en) * 2022-04-01 2023-10-24 上海明悦医疗科技有限公司 Primer combination product, kit for quality detection and genome quality detection method
CN116769879A (en) * 2023-07-07 2023-09-19 中信湘雅生殖与遗传专科医院有限公司 Nucleic acid products, kits and methods for quality control of MDA amplification products

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
DNA Degradation Test Predicts Success in Whole- Genome Amplification from Diverse Clinical Samples;Fengfei Wang等;《Journal of Molecular Diagnostics》;20070930;第9卷(第4期);第441-451页 *
Using Multiplex PCR for Assessing the Quality of Whole Genome Amplifi ed DNA;Amin El-Heliebi等;《Whole Genome Amplification》;20151231;第1347卷;第119-128页 *

Also Published As

Publication number Publication date
CN118421769A (en) 2024-08-02

Similar Documents

Publication Publication Date Title
JP7256748B2 (en) Methods for targeted nucleic acid sequence enrichment with application to error-corrected nucleic acid sequencing
CN118421769B (en) Multiplex PCR primer group for detecting quality of samples used for second-generation sequencing and application thereof
CN103305625B (en) Method and kit for detecting non-small cell lung cancer drive gene mutation spectrum, and application
CN110964814B (en) Primers, compositions and methods for nucleic acid sequence variation detection
CN105229175A (en) For increasing and measuring the method for RNA fusion gene variant, the method distinguishing them and relevant primer, probe and test kit
KR102006803B1 (en) A Method for Multiple Detection of Methylated DNA
CN102586420B (en) Method and kit for assaying breast cancer susceptibility genes
CN104745679A (en) Method and kit for non-invasive detection of EGFR (epidermal growth factor receptor) gene mutation
CN114606299A (en) Method and kit for improving the sensitivity of digital PCR
CN105256057A (en) Colon cancer microsatellite instability detection kit based on next generation sequencing platform
CN102206702A (en) miR-122 (micro-ribonucleic acid-122) as serum marker for liver diseases
KR102098773B1 (en) Primers for distinguishing Anguilla japonica from other species such as A. bicolor, A. rostrata, A. anguilla and A. marmorata and detection method using Real-time PCR
EP4043585A1 (en) Method for determining if origin of biological sample is from liver tissue
WO2023193456A1 (en) Biological composition, method for preparing same, and use thereof
Senst et al. Application of the Agilent 2100 Bioanalyzer instrument as quality control for next‐generation sequencing
JP6544783B1 (en) Probe / primer library for cancer diagnosis
CN109234370B (en) MUT gene mutation detection kit
CA2935789A1 (en) Lung cancer determinations using mirna
US20220049313A1 (en) Primer for detecting tumor microsatellite instability, use thereof and kit
KR101775953B1 (en) Detection methods of mutation and the kits
KR20240118676A (en) Composition for predicting a risk of developing liver cancer
US20240102089A1 (en) Method for Evaluating Adapter Ligation Efficiency in Sequencing of DNA Sample
US20170159125A1 (en) Micro-rna biomarkers for haemolysis and methods of using same
CN112522369A (en) Method for designing Blocker double chain of ARMS-TaqMan Blocker system
CN113337586A (en) Digital PCR reaction system for detecting BRAF V600E locus and application thereof

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
GR01 Patent grant
GR01 Patent grant