CN110699455A - Human circulating tumor cell MSI detection primer group, kit and detection method - Google Patents
Human circulating tumor cell MSI detection primer group, kit and detection method Download PDFInfo
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Abstract
The invention discloses a primer group, a kit and a detection method for detecting human circulating tumor cell MSI, wherein the primer group has high sensitivity and strong specificity when detecting human MSI state; the kit can quickly, efficiently and accurately detect the MSI state of the human circulating tumor cells; the detection method has the advantages of real and reliable result, strong consistency among rooms, easy popularization and the like. The invention optimizes the circulating tumor cell detection, and has high sensitivity and accuracy of the detection result on the basis of realizing non-wound detection.
Description
Technical Field
The invention relates to the field of gene detection, in particular to a primer group, a kit and a detection method for detecting MSI of human circulating tumor cells.
Background
Circulating Tumor Cells (CTCs) refer to various types of tumor cells that have left the primary site and the metastatic site of a tumor and have entered the blood circulation. CTC has obvious advantages in the aspects of early tumor discovery, medication guidance, relapse detection after prognosis, tumor metastasis prompting and the like: 1) CTCs are meaningful for early diagnosis of malignant tumors, and research shows that a small amount of CTCs can be detected in peripheral blood of an early asymptomatic malignant tumor patient, while CTCs are not detected in blood of a benign disease and a healthy examinee; 2) the CTCs and tumor cells in primary foci have similar genetic genetics characteristics, so that noninvasive and sustainable monitoring of tumor course can be realized, and medication can be guided; 3) similar to early tumor discovery, prognosis can continuously monitor recurrence status; 4) CTC is used as a bridge between a primary focus and a metastatic focus, carries a large amount of information of tumor progression and metastasis, has extremely strong metastatic potential, and plays an important role in tumor metastasis and recurrence.
Microsatellites (microsatellites) are short tandem repeats distributed throughout the human genome. Microsatellite instability (MSI) is caused by a defect in the occurrence of the mismatch repair (MMR) gene and is closely associated with tumorigenesis. It is shown that the length of the microsatellite in the tumor cell is increased or shortened as compared with that in the normal cell due to the insertion or deletion of the repeating unit.
There are currently two approaches to detecting MSI in tumor cells: 1) molecular level: including multiplex PCR-capillary electrophoresis, NGS; 2) and (4) immunohistochemistry. Among them, the multiplex fluorescence PCR-capillary electrophoresis method for detecting MSI state is an internationally recognized gold standard, and is jointly recommended by international well-known institutions such as the national center for comprehensive cancer (NCCN) and ASCO. In addition, the multiplex PCR-capillary electrophoresis has certain advantages in high-throughput detection.
On 23.5.2017, the PD-1 antibody drug Keytruda was first approved by the FDA for the treatment of solid tumors of the dMMR/MSI-H type, an anti-tumor therapy that was differentiated only by molecular markers. It is indicated that patients with solid tumors characterized by the type dMMR/MSI-H benefit more after immunosuppression by Keytruda. Patients with solid tumors of MSI-H were clinically distinguished by tumor microsatellite instability (MSI) detection. However, there is currently no data in China that indicates more accurately which loci Panel will indicate MSI status.
Disclosure of Invention
Aiming at the problems, the invention develops the primer group, the kit and the detection method for detecting the MSI of the human circulating tumor cells, optimizes the circulating tumor cell detection, and has high sensitivity and accuracy of detection results on the basis of realizing non-wound detection.
In order to achieve the purpose, the invention adopts the technical scheme that:
the invention provides a primer group for detecting MSI of human circulating tumor cells, which comprises 5 pairs of fluorescence labeling primers for detecting 5 MSI representative sites BAT-25, BAT-26, D2S123, D5S346 and D17S250, wherein the primers and fluorescence labels thereof are as follows:
preferably, the primer set further comprises 2 pairs of fluorescence labeled primers of 2 internal standard sites PentaC and PentaE, and the primers and the fluorescence labels thereof are as follows:
the invention also provides a human circulating tumor cell MSI detection kit, which comprises the detection primer group.
Preferably, the detection kit further comprises a MultiPlex buffer and a MultiPlex DNA polymerase.
Preferably, the test kit further comprises an internal reference marker (e.g., DDLIZE500) and a negative control sample (e.g., K562 gDNA).
The invention also provides a method for detecting the MSI of the human circulating tumor cells, which comprises the following steps:
1) dispensing an amplification mixture to the bottom of the PCR reaction tube and adding CTCs sample, negative control sample and negative control respectively, wherein the amplification mixture comprises Multiplex buffer solution, MSI primer mixture, Multiplex DNA polymerase and NFH2O, the number of the CTCs in the CTCs sample is not less than 20/mu l;
2) transferring the PCR reaction tube to a PCR instrument for PCR amplification, wherein the amplification conditions are as follows:
3) preparing a sample to be analyzed, and carrying out capillary electrophoresis detection on a PCR amplification product;
4) analyzing the data output in step 3) to determine the MSI status of the test sample.
Preferably, the CTCs sample is obtained in step 1) by: centrifuging whole blood of tumor patient to separate plasma and leukocyte, resuspending leukocyte with PBS, and separating
The system was enriched for CTCs and resuspended in not less than 20 CTCs with 1. mu.l PBS.
Preferably, the internal reference marker DDLIZE500 is used in step 3) to indicate fragment lengths within 35-500 bp.
Preferably, Applied is used in step 3)
3500Dx gene analyzer was used for capillary electrophoresis detection.
Preferably, used in step 4)
And 5, analyzing the data output in the step 3) by software.
The invention has the following beneficial effects:
the invention provides a primer and a probe for detecting MSI state of human circulating tumor cells, which comprise 7 pairs of fluorescence labeling primers, wherein 5 of the fluorescence labeling primers are MSI detection representative sites jointly recommended by International known institutions such as NCCN, ASCO and the like, 2 of the fluorescence labeling primers are internal standard sites, and 5 of the fluorescence labeling primers comprise two mononucleotide repeat sequence sites (BAT-25 and BAT-26), 3 dinucleotide repeat sequence sites (D2S123, D5S346 and D17S250), and 2 pentanucleotide repeat sequence internal standard sites PentaC and Penta E. The primer and the probe have high sensitivity and strong specificity when detecting the human MSI state.
The invention provides a kit for detecting MSI state of human circulating tumor cells, wherein the kit provides a matched internal reference marker DDLIZE500 which indicates the fragment length within 35-500 bp; the invention also provides a negative control sample, gDNA extracted from the K562 cell line is diluted to 50 pg/mu l to be used as the negative control sample of the detection kit, the negative control sample is added in each batch of detection, and the negative control (water control) is arranged to check the accuracy of the detection batch. The kit can rapidly, efficiently and accurately detect the MSI state of the human circulating tumor cells.
Thirdly, the invention provides a detection method for detecting MSI state of human circulating tumor cells, which optimizes a direct expansion system, does not need extraction, can realize complete detection by adding processed CTCs and contrast white blood cells according to requirements, the traditional tumor tissue detection initial amount is 2ng, the invention can obtain a detection result with high sensitivity and high accuracy from CTCs of 50pg or more, and the invention is suitable for Applied authenticated type
3500Dx gene analyzer, output data can be used
Software 5 (applied biosystems) to determine the MSI status of the test samples. In addition, the present invention provides for analyzing relevant Panels text files, allowing for the use of
The genotype is automatically allocated by software, so that the method has wider application foundation, more sufficient related research data and easier finding of research basis for medication guidance. The detection method has the advantages of simple operation, high sensitivity, high specificity, real and reliable result, strong consistency among rooms, easy popularization and the like.
Drawings
Fig. 1 is a reference example of ddlife 500.
Fig. 2 is an example of an MSS clinical sample in an embodiment of the present invention.
FIG. 3 is an example of a negative control sample in an embodiment of the present invention.
FIG. 4 an example of a clinical sample of MSI-L in an embodiment of the present invention.
FIG. 5 an example of clinical samples of MSI-H in an embodiment of the present invention.
Detailed Description
For a better understanding of the present invention, the contents of the present invention will be further explained below with reference to the drawings and examples, but the present invention is not limited to the following examples.
Examples
Instrument and reagent
(I) apparatus
1. PCR instrument
Bio-RAD C100096 well gradient augmentor (1851197).
2、
System for controlling a power supply
At present, the number of the current day,
the only product that has been approved for marketing by the FDA is the system that uses immunomagnetic bead enrichment of EpCAM antibodies, which are tumor cell epithelial markers, to separate CTCs from circulating blood in a forward enrichment manner.
3、ABI
3500Dx genetic analyzer
(II) reagent
1. PBS 1L formulation ph 7.4:
potassium dihydrogen phosphate (KH)2PO4)：0.24g，
Disodium hydrogen phosphate (Na)2HPO4)：1.44g，
Sodium chloride (NaCl): 8g of the total weight of the mixture is 8g,
potassium chloride (KCl): 0.2g of a mineral oil in the form of a mineral oil,
adding deionized water about 800mL, stirring thoroughly to dissolve, adding concentrated hydrochloric acid to adjust pH to 7.4, and adding volume to 1L.
2. The invention relates to primers, probe sequences and fluorescence labeling information as listed in the following table:
| BAT-25-F | GCATGGCTTTCCTCGCCTCC | 6-FAM |
| BAT-25-R | GACATTCTGCATTTAACTATG | |
| BAT-26-F | GCAGCAGTCAGAGCCCTTAAC | VIC |
| BAT-26-R | TTAACCCTCTGGCCTAGGGA | |
| D2S123-F | CTGCCTTTAACACTGCTATT | VIC |
| D2S123-R | TTTGAATTGGAGGGGACTTTC | |
| D5S346-F | CTTGACAATAGACAAATATG | 6-FAM |
| D5S346-R | TCGAATGGAGATTGGCCTG | |
| D17S250-F | AGCCACTCAGCTGGCCATATAT | 6-FAM |
| D17S250-R | GTAAGCATAAAAAGGAAGAATC | |
| Penta C-F | GAGAGCATTCCAACACTGAG | VIC |
| Penta C-R | CTGGTAATAGAAAAGAAGGGA | |
| Penta E-F | GGGTTATTAATTGAGAAAAC | TAMRA |
| Penta E-R | TGTGGTGGTAGGCACCTGTAAT |
3. the reagent used for PCR amplification in the invention comprises two parts:
the Pre-PCR part comprises the following components:
the Post-PCR part comprises the following components:
note: 10 × MSI Primer Mix was made by mixing equal amounts of primers.
2 XMultiplex Buffer, Multiplex DNA Polymerase from TaKaRa MultiplexxPCR Assay Kit.
Second, Experimental methods
In the present invention, data analysis requires5 analysis software. The corresponding analysis parameters were selected according to the instrument instructions to generate an electropherogram with a data sheet showing the PCR fragment length (in nt) and peak height (in RFU). And outputting a detection result text after data analysis.
The following experiments used an internal standard, ddlife 500, as shown in fig. 1, occupying the orange fluorescence channel. Detection was performed using 4-color fluorescence: blue, green, yellow, orange, etc. 4 color channels.
(first) negative sample experiment
1. Separating and obtaining CTCs collecting fluid sample
Take circulating tumor cells of a conventional MSS tumor patient (from Suzhou chem's institute of medicine). Taking at least 10ml of whole blood of a tumor patient, centrifuging at 6000rpm for 10 minutes, and separating plasma and white blood cells. The leukocytes were resuspended in 5ml PBS; for blood plasma
The system is enriched with CTCs, and requires no less than 20 CTCs and 1 μ l PBS to resuspend the cells.
2. PCR amplification
1) 2 XMultiplex Buffer, 10 XMSI Primer Mix were thawed at 4 ℃ and the reagents were vortexed for 5-10 seconds prior to use.
2) Amplification mixtures were prepared and 0.1-2 reactions were added to the preparation of the amplification mixtures to compensate for losses during the tube rotation.
3) Amplification mixture:
| 2×MultiPlex Buffer | 5μl |
| 10×MSI Primer Mix | 1μl |
| MultiPlex DNA Polymerase | 0.2μl |
| NFH2O | 2.8μl |
| Mix | 9μl |
note: too small a volume is prone to pipetting inaccuracies and it is recommended to formulate the amplification mixture to ensure pipetting accuracy and uniformity of the individual reaction tubes.
4) Clearly marking each PCR reaction tube, and subpackaging the amplification mixture to the bottom of each marked PCR reaction tube.
5) And adding the CTCs collected liquid sample obtained by separation to the bottom of the PCR reaction tube.
6) A negative control sample and a negative control were prepared by adding 1. mu.l each of K562DNA (50 pg/. mu.l) and NFH 2O.
7) Transferring the PCR tube added in the PCR reaction system to a PCR instrument, wherein the reaction conditions are as follows:
if a sample with difficult amplification occurs, the number of amplification cycles can be increased appropriately, but not more than 45 cycles. After amplification was complete, the amplified product was stored at 4 ℃.
3. Analysis of amplification products
1) Thawing Hi-Di in ice bathTMFormamid (commercially available) and DDLIZE500, these reagents were vortexed for 5-10 seconds prior to use.
2) Amplification mixtures were prepared and 0.1-2 reactions were added to the preparation of fragment analysis mixtures to compensate for losses during the tube rotation.
3) Fragment analysis mixture:
| Hi-DiTM Formamid | 8.8 |
| DD LIZE | |
| 500 | 0.2μl |
| Mix | 9μl |
4) and arranging a product reaction plate, and subpackaging the fragment analysis mixture to the bottom of each arranged PCR reaction tube.
5) Add 1. mu.l of PCR product to the bottom of the reaction tube. The reaction solution was collected by centrifugation at 1000rpm for 10 seconds to the bottom of the tube.
6) The sample was denatured by heating at 95 ℃ for 3 minutes and then immediately cooled on crushed ice or in ice water for 3 minutes. The samples were denatured prior to loading.
The results of the detection are shown in FIG. 2. No unstable sites were found.
(II) negative control sample experiment
1. Cell harvesting
Cultured K562 cells (purchased from ATCC) were collected. The QIAamp DNA Mini Kit (Qiagen) was selected and genomic DNA was extracted as described. The extracted DNA concentration was determined with a Qubit and diluted to 50 pg/. mu.l for subsequent detection.
2. PCR amplification
1) 2 XMultiplex Buffer, 10 XMSI Primer Mix were thawed at 4 ℃ and the reagents were vortexed for 5-10 seconds prior to use.
2) The amplification mixture was prepared for 6 reactions. To compensate for losses during the tube rotation, 0.2 reactions were added when formulating the amplification mix.
3) Amplification mixture:
| 2×MultiPlex Buffer | 5μl |
| 10×MSI Primer Mix | 1μl |
| MultiPlex DNA Polymerase | 0.2μl |
| NFH2O | 2.8μl |
| Mix | 9μl |
4) clearly marking each PCR reaction tube, and subpackaging the amplification mixture to the bottom of each marked PCR reaction tube.
5) K562DNA (3 repeats), H, diluted to 50 pg/. mu.l2O (3 replicates), 1. mu.l each was added to the bottom of the PCR reaction tube.
6) Transferring the PCR tube added in the PCR reaction system to a Bio-RAD C100096-hole gradient amplification instrument under the following reaction conditions:
| 1 | 95℃ | 5min |
| 2 | 95℃ | 30s |
| 3 | 61℃ | 90s |
| 4 | 72℃ | 30s |
| 5 | Goto 2 | For 35times |
| 6 | 72℃ | 15min |
| 7 | 4-12℃ | Hold |
3. analysis of amplification products
1) Thawing Hi-Di in ice bathTMFormamid and DD LIZE500, these reagents were vortexed for 5-10 seconds prior to use.
2) Amplification mixtures were prepared and to compensate for losses during the tube rotation, 0.2 reactions were added when formulating the fragment analysis mixtures.
3) Fragment analysis mixture:
| Hi-DiTM Formamid | 8.8 |
| DD LIZE | |
| 500 | 0.2μl |
| Mix | 9μl |
4) the product reaction plates were arranged and the fragment analysis mixtures were dispensed to the bottom of each arranged PCR reaction tube, 9. mu.l per reaction tube.
5) Add 1. mu.l of PCR product to the bottom of the reaction tube. The reaction solution was collected by centrifugation at 1000rpm for 10 seconds to the bottom of the tube.
6) The sample was denatured by heating at 95 ℃ for 3 minutes and then immediately cooled on crushed ice or in ice water for 3 minutes. The samples were denatured prior to loading.
The results of the detection are shown in FIG. 3. No unstable sites were found.
(III) Positive clinical sample experiment
1. Circulating tumor cells of conventional MSI-L tumor patients and circulating tumor cells of MSI-H tumor patients (from Shinzea chekiangen medical laboratory) are taken as examples. Taking at least 10ml of whole blood of a tumor patient, and carrying out 6000g of centrifugation to separate blood plasma and white blood cells. The leukocytes were resuspended in 5ml PBS; for blood plasma
The system is enriched with CTCs, and requires no less than 20 CTCs and 1 μ l PBS to resuspend the cells.
2. PCR amplification
1) 2 XMultiplex Buffer, 10 XMSI Primer Mix were thawed at 4 ℃ and the reagents were vortexed for 5-10 seconds prior to use.
2) Amplification mixtures were prepared for 6 reactions. To compensate for losses during the tube rotation, 0.2 reactions were added when formulating the amplification mix.
3) Amplification mixture:
| 2×MultiPlex Buffer | 5μl |
| 10×MSI Primer Mix | 1μl |
| MultiPlex DNA Polymerase | 0.2μl |
| NFH2O | 2.8μl |
| Mix | 9μl |
4) clearly marking each PCR reaction tube, and subpackaging the amplification mixture to the bottom of each marked PCR reaction tube.
5) Circulating tumor cells and corresponding leukocyte resuspension (2 replicates) of tumor patients were added to the bottom of the PCR reaction tube in an amount of 1. mu.l each.
6) Each of K562gDNA (50 pg/. mu.l) and NFH2O (1. mu.l) was added as a negative control sample and a negative control.
7) Transferring the PCR tube added in the PCR reaction system to a Bio-RAD C100096-hole gradient amplification instrument under the following reaction conditions:
3. analysis of amplification products
1) Thawing Hi-Di in ice bathTMFormamid and DD LIZE500, these reagents were vortexed for 5-10 seconds prior to use.
2) Amplification mixtures were prepared for 6 reactions. To compensate for losses during the tube transfer, 0.2 reactions were added when formulating the fragment analysis mixtures.
3) Fragment analysis mixture:
| Hi-Di Formamid | 8.8 |
| DD LIZE | |
| 500 | 0.2μl |
| Mix | 9μl |
4) the product reaction plates were arranged and the fragment analysis mixtures were dispensed to the bottom of each arranged PCR reaction tube, 9. mu.l per reaction tube.
5) Add 1. mu.l of PCR product to the bottom of the reaction tube. The reaction solution was collected by centrifugation at 1000rpm for 10 seconds to the bottom of the tube.
6) The sample was denatured by heating at 95 ℃ for 3 minutes and then immediately cooled on crushed ice or in ice water for 3 minutes. The samples were denatured prior to loading.
The detection result of clinical MSI-L is shown in FIG. 4, only BAT-26 site appears unstable and belongs to MSI-L; the results of clinical MSI-H assays are shown in FIG. 5, where all loci (BAT-25, BAT-26, D2S123, D5S346, D17S250) were unstable, and are typical of MSI-H assays.
It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.
Claims (10)
1. The primer group for detecting the MSI of the human circulating tumor cells comprises 5 pairs of fluorescence labeled primers for detecting 5 MSI representative sites BAT-25, BAT-26, D2S123, D5S346 and D17S250, wherein the primers and fluorescence labels thereof are as follows:
2. the human circulating tumor cell MSI detection primer set of claim 1, further comprising 2 pairs of fluorescence labeled primers of 2 internal standard sites Penta C and Penta E, wherein the primers and their fluorescence labels are as follows:
3. a human circulating tumor cell MSI detection kit comprising the human circulating tumor cell MSI detection primer set of claim 1 or 2.
4. The human circulating tumor cell MSI detection kit of claim 3, in which the detection kit further comprises MultiPlex buffer, MultiPlex DNA polymerase.
5. The human circulating tumor cell MSI detection kit of claim 4, in which the detection kit further comprises an internal reference marker and a negative control sample.
6. The detection method of the MSI of the human circulating tumor cells comprises the following steps:
1) dispensing an amplification mixture into the bottom of the PCR reaction tube and adding CTCs samples, negative control samples and negative controls respectively, wherein the amplification mixture comprises Multiplex buffer solution, MSI primer mixture, Multiplex DNA polymerase and NFH2O, the number of the CTCs in the CTCs sample is not less than 20/mu l;
2) transferring the PCR reaction tube to a PCR instrument for PCR amplification, wherein the amplification conditions are as follows:
3) preparing a sample to be analyzed, and carrying out capillary electrophoresis detection on a PCR amplification product;
4) analyzing the data output in step 3) to determine the MSI status of the test sample.
7. The method for MSI detection of circulating human tumor cells of claim 6, wherein the CTCs are obtained from step 1) by: centrifuging whole blood of tumor patient to separate plasma and leukocyte, resuspending leukocyte with PBS, and separating
The system was enriched for CTCs and resuspended in not less than 20 CTCs with 1. mu.l PBS.
8. The method for detecting MSI in human circulating tumor cells of claim 6, wherein the fragment length within 35-500bp is indicated by using the internal reference marker DD LIZE500 in step 3).
9. The method for MSI detection of circulating human tumor cells of claim 6, wherein the step 3) is performed by using Applied
3500Dx gene analyzer was used for capillary electrophoresis detection.
10. The method for MSI detection of circulating human tumor cells of claim 9, wherein the MSI detection is performed in step 4)
And 5, analyzing the data output in the step 3) by software.
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