CN116640844A - Differentiation type thyroid cancer circulating tumor cell detection kit - Google Patents

Differentiation type thyroid cancer circulating tumor cell detection kit Download PDF

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Publication number
CN116640844A
CN116640844A CN202210136153.3A CN202210136153A CN116640844A CN 116640844 A CN116640844 A CN 116640844A CN 202210136153 A CN202210136153 A CN 202210136153A CN 116640844 A CN116640844 A CN 116640844A
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artificial sequence
kit
circulating tumor
thyroid cancer
dna
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柳卫
付煜
葛世彬
孙闯
郑倩
黄继萍
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Jiangsu Province Hospital First Affiliated Hospital Of Nanjing Medical University
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Jiangsu Province Hospital First Affiliated Hospital Of Nanjing Medical University
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Abstract

The invention discloses a differentiated thyroid cancer circulating tumor cell detection kit. The kit comprises an 8 μm nanofiltration membrane and the Tg high specificity mRNA nucleic acid probe composition of claim 1. The kit provided by the invention can be used for detecting the CTC with the specificity of DTC, so that more accurate clinical data related to thyroid patient prognosis and recurrence risk assessment can be provided for clinicians. In addition, the DTC specific CTC integrated detection kit has the advantages of high accuracy, high sensitivity, low cost, simplicity and convenience in operation, difficulty in pollution by other nucleic acid components and the like.

Description

Differentiation type thyroid cancer circulating tumor cell detection kit
Technical Field
The invention belongs to the field of biological detection, and relates to a differentiated thyroid cancer circulating tumor cell detection kit.
Background
Thyroid cancer (Thyroid cancer) is the most common malignancy of the endocrine system, and its incidence has increased significantly in the last 20 years, and is domestic and ranked at 7 th of malignancy, and at 4 th of female cancer patients. Differentiated thyroid cancers (Differentiated thyroid cancer, DTC) include papillary and follicular adenocarcinomas, accounting for 90% of thyroid cancers. Although DTC patients have a good overall prognosis, with a survival rate of 90% over 10 years, if DTC is distant from metastasis, the quality of life of the patient will be severely reduced, and the survival rate will be less than 50% over 5 years. 131 Iodine therapy is one of the effective treatments for recurrence/metastasis in DTC patients, but about 30% of patients are refractory to iodine DTC (radio-refractory DTC, RR-DTC) and the 10-year survival rate of patients is less than 10%.
Ultrasonic guided fine needle aspiration histological biopsy (Ultrasound (US) -guided fine needle aspiration biopsy, US-guided FNA) can perform cell morphology analysis on DTC specimens to identify benign or malignant lesions of thyroid, which is a "gold standard" recommended by current european DTC diagnostic guidelines, but cannot evaluate disease states such as recurrence, metastasis and whether or not it is RR-DTC. Thyroglobulin (Tg) is currently widely used in the diagnosis and treatment of clinical thyroid cancer, and is a reliable indicator for predicting whether thyroid cancer is distant metastasis and evaluating the risk of recurrence after DTC surgery. However, the detection level of Tg in some patients is often affected by the detection level of Tg antibodies (tgabs); in addition, tg is of little value for prognosis in RR-DTC patients. Therefore, a new tumor marker is needed in clinic to assist in judging whether the DTC patient has metastasis or not and evaluating the treatment prognosis of the DTC patient.
The circulating tumor cells (Circulating tumor cell, CTCs) are tumor cells that have been released from the primary focus or metastasis of a solid tumor and entered into the peripheral blood circulation due to spontaneous or clinical procedures. A large number of researches show that the number of CTCs is closely related to prognosis of various tumor patients such as colon cancer, breast cancer, non-small cell lung cancer and prostate cancer. For DTC, some distant metastases were developed and the number of CTCs in RR-DTC patients was increased. Therefore, CTC detection is expected to play a role in DTC patient prognosis. However, CTCs are extremely contained in bloodLess, at 10 6 ～10 7 Only 1 CTC can be detected in individual blood cells, so that the detection of CTCs has high requirements on the sensitivity and specificity of the detection technology. Currently, the detection process of CTCs mainly includes separation and enrichment of CTCs and identification of CTCs. The enrichment method of CTC mainly comprises gradient centrifugation, filtration, immunomagnetic separation and the like; the filtering membrane method is a non-specific method for separating tumor cells according to the fact that tumor cells are generally larger than blood cells, cells with diameters larger than 8 mu m including tumor cells can be obtained through filtering, the obtained tumor cells are complete in morphology and good in cell property, and the method is suitable for subsequent further detection and analysis. Current CTC identification methods include immunological techniques, reverse transcription polymerase chain reaction techniques, enzyme linked immunosorbent assay techniques, CTC chip techniques, etc. However, the above CTC detection method is mainly directed to a non-specific antigen or nucleic acid expressed by tumor cells, and it is not possible to determine the origin of tumor tissue of CTCs, which brings about various uncertain factors for evaluation of tumor therapeutic effects. Therefore, the establishment of a detection method of the CTC derived from the thyroid tissue, which has high accuracy, high sensitivity, simplicity, easiness and low cost, plays a very important role in clinical thyroid cancer disease management.
Foreign scholars designed specific probes for Tg highly expressed in thyroid cells, and detected CTC enriched in thyroid cancer patients by using a fluorescence quantitative PCR method (Quantitative real-time PCR, qRT-PCR) with a positive rate of 39.6% (19/48), respectively; however, the qRT-PCR method used in the study is complicated in operation, high in cost, and susceptible to factors such as nucleic acid contamination, and needs to perform RNA extraction and reverse transcription on each enriched CTC, and the accuracy of the result is easily affected by the RNA yield, which may also be a cause of low Tg-specific CTC detection positive rate.
Disclosure of Invention
The invention aims at overcoming the defects of the prior art and providing a differentiated thyroid cancer circulating tumor cell detection kit.
It is another object of the present invention to provide a method for detecting circulating tumor cells of thyroid cancer which is not aimed at disease diagnosis.
The aim of the invention can be achieved by the following technical scheme:
a Tg high specificity mRNA nucleic acid probe composition consists of 48 probes shown in SEQ ID NO.1-SEQ ID NO. 48.
The invention relates to application of a Tg high specificity mRNA nucleic acid probe composition in preparation of a differentiated thyroid cancer circulating tumor cell detection kit.
A differentiation type thyroid cancer circulating tumor cell detection kit comprises a nanofiltration membrane with the diameter of 8 mu m and the Tg high-specificity mRNA nucleic acid probe composition.
Preferably, the kit further comprises relevant reagents for solidifying, hybridizing and washing the enriched cells.
The invention discloses a method for detecting thyroid cancer circulating tumor cells, which does not aim at disease diagnosis.
As a preferred aspect of the present invention, the method comprises the steps of:
(1) Enriching the circulating tumor cells in the blood sample using an 8 μm nanofiltration membrane;
(2) Solidifying the enriched cells, hybridizing and washing by using the Tg high specificity mRNA nucleic acid probe composition, and counting the number of CTCs in blood under a fluorescence microscope.
The beneficial effects are that:
the invention provides a DTC specific CTC integrated detection kit, which can accurately count the number of CTCs derived from a DTC patient in vitro so as to evaluate the relativity of prognosis and recurrence of the DTC patient. The kit provided by the invention can be used for detecting the CTC with the specificity of DTC, so that more accurate clinical data related to thyroid patient prognosis and recurrence risk assessment can be provided for clinicians. In addition, the DTC specific CTC integrated detection kit has the advantages of high accuracy, high sensitivity, simplicity and convenience in operation, difficulty in pollution by other nucleic acid components and the like. Successful development of this technology or product will maximally meet the urgent market and clinical demands for molecular diagnosis specific to thyroid cancer. In view of the defects of detection of CTC in serum of thyroid cancer patients in the current market, the DTC-specific CTC integrated detection kit established by the research has good market application prospect and is expected to generate higher economic benefit and social benefit.
Drawings
FIG. 1 shows a test flow of the kit
FIG. 2 specificity verification of the present kit
FIG. 3 clinical diagnostic efficacy of the kit
Detailed Description
Example 1
The detection of thyroid cancer circulating tumor cells by the kit CTC is divided into two steps (figure 1):
1. enrichment of CTCs: the circulating tumor cells in 5ml fresh blood samples were enriched using an 8 μm nanofiltration membrane, and the steps were performed as follows:
(1) Sample requirement
a) The venous blood sample is collected by using a vacuum anticoagulation blood collection tube, and the blood collection tube is slightly and reversely mixed for 8 times in time after collection.
b) To ensure sample quality, if the subject has no other blood collection items, 1mL of venous blood is firstly collected by using a 2mL anticoagulation tube, and the tube is discarded and not used as a formal sample; and collecting a test sample by using the BD EDTA anticoagulant tube, wherein the sample size is ensured to be at least 5mL. If the subject has other blood collection items, the sample required by the test is collected after the other blood collection items are finished, so that the step of collecting the first tube of blood can be omitted.
c) The sample is stored without refrigeration and at room temperature, and the pretreatment is required to be carried out within 2 hours after the sample is collected. If the internal cross-over detection cannot be carried out within 2 hours, the test paper is vertically placed in a refrigerator at the temperature of 4 ℃ for preservation, and the preservation time cannot exceed 24 hours. The samples stored in a refrigerator at 4℃were rewarmed for at least 30 minutes at room temperature 7 prior to processing.
(2) Filter rinse
a) The filter up and down plugs were opened and the filter was placed on a rinse bracket.
b) Approximately 2mL of 75% medical alcohol was added to the filter using a pasteur tube until it was naturally filtered out of the lower mouth of the filter.
c) 4mL of physiological saline is added into the filter for rinsing, residual alcohol in the filter is removed, and the physiological saline is naturally filtered.
d) And c), repeating the step c), reserving a proper amount of physiological saline to ensure that the filter membrane is wet, and installing a filter and plugging. Note that: if there is a condition that alcohol cannot be naturally filtered out or is too slow to be filtered out during the filter rinsing process, the filter is immediately discarded.
(3) Sample pretreatment: 375. Mu.L of 8% PFA was added to a 15mL centrifuge tube, and 3mL of physiological saline was added. 5mL of blood sample was taken and added to a 15mL centrifuge tube (to 8 mL). Physiological saline was added to 15mL, and the mixture was gently inverted and mixed for 8 times, and left to stand at room temperature for 10min, to pre-fix the cells. When multiple blood samples exist, the blood samples should be treated in one part, so that cross contamination is avoided and the fixing agent is forgotten to be added.
(4) Sample separation
a) The filter was placed in the middle of the a10 support module to ensure that the lower plug was pierced by the lower needle.
b) About 8mL of the fixed sample is added into the filter by using a pasteurization tube, the start is clicked, after the sample separation is finished, the rest 7mL of the sample is added into the filter by using the pasteurization tube, and the start is clicked again, so that the sample separation is finished.
c) 4mL of PBS was added to the filter tube, and the sample was rinsed by clicking on "Start".
d) Repeating step c) 2 times.
e) Methanol fixation: 500. Mu.L of methanol was added to the filter, and after 1min at room temperature, the "start" button was clicked to run the apparatus to drain the methanol. If methanol remains, the filter is taken down from the machine, the lower rubber plug is opened, and the upper rubber plug is covered, so that the residual methanol naturally flows out.
f) Film taking and film sticking: removing the filter, taking out the filter membrane, dripping 5 mu L of methanol into the left upper corner of the glass slide, sticking the filter membrane to the glass slide with the right side facing upwards, and baking for 10min at 37 ℃.
g) Removing cells on the back of the filter membrane: and (3) dropwise adding 10 mu L of deionized water on the glass slide, tearing off the baked filter membrane by using tweezers, then flatly attaching the glass slide with the dropwise added deionized water, flatly attaching as much as possible, and naturally airing for 10min for later detection.
2. CTC detection: after a series of treatments such as fixed-line, tg probe hybridization and washing are performed on the enriched cells, the number of CTCs in the blood is counted under a fluorescence microscope.
(1) Immobilization and permeabilization of CTC cells
a) The filters were placed in 12-well plates.
b) Washing with 1mL of 1 XPBS
c) 1mL of methanol-acetic acid (MeOH-AcOH) fixative was added to fix and permeabilize the cells.
d) Incubation at room temperature for 10min
e) Until 48 hours before hybridization, cells were stored in MeOH-AcOH at 2-8deg.C.
(2) FISH hybridization
a) MeOH-AcOH was pipetted off the 12-well plate.
b) 1mL of buffer A was added and then incubated at room temperature for 2-5 minutes.
c) Assembling a wet box: 150mm tissue culture plate; the bottom is evenly spread with the paper towel fully absorbing water and a single layer is placed above the paper towelHelping to prevent evaporation of probe solution from under the coverslip.
d) In a humidified box, 100. Mu.L of hybridization buffer containing probes is dispensed onto a paraffin membrane.
e) The coverslip was gently tilted with the cell side down and transferred to 100. Mu.L of hybridization buffer droplets containing probes.
f) The humidification chamber is covered and sealed with a paraffin membrane.
g) Incubate at 37℃for 2 hours in the absence of light. (incubation may last up to 16 hours).
h) The coverslip was gently tilted, cell side up, and transferred to a new 12-well plate containing 1mL of buffer a.
i) Incubate at 37℃for 30 min in the absence of light.
j) After aspiration of buffer A, 1mL of DAPI nuclear dye (DAPI in buffer A, final concentration 5 ng/mL) was added to counterstain the nuclei.
k) Incubate at 37℃for 30 min in the absence of light.
l) aspiration of DAPI staining buffer followed by addition of 1mL of buffer B. Incubate at room temperature for 2-5 minutes.
m) a small drop (about 15 μl) of anti-fluorescence quenching caplet was added to the microscope slide and the coverslip was covered onto the slide with the cell side down.
n) gently remove excess coverslip from the periphery of the coverslip.
o) sealing the perimeter of the cover slip with clear nail polish and allowing it to dry.
p) if necessary, gently wipe the salt components that have been dried off on the coverslip with water.
q) imaging.
Example 2 specificity verification
First, the specificity of the kit was verified by using the thyroid papillary carcinoma cell line B-CPAP, the follicular carcinoma cell line FTC-133 and the thyroid undifferentiated carcinoma cell line HTH-74, and the detection method was the same as in example 1. As a result, it was found (FIG. 2) that the Tg-mRNA FISH probe used in the present kit can detect the Tg-mRNA fluorescence signal (red) in the cytoplasm of DTC cell line (B-CPAP, FTC-133) without showing the Tg-mRNA fluorescence signal in the cytoplasm of ATC cell line (HTH-74), and only the nucleus was stained blue with DAPI. Then, 10 cases of differentiated thyroid cancer metastasis were collected and detected by using the Tg-mRNA FISH probe of the kit. As a result, it was found that specific red fluorescence was detected by CTCs derived from thyroid in thyroid cancer patients, while nuclei were stained blue by DAPI; however, since leukocytes around CTCs do not express Tg-mRNA, no fluorescent signal of Tg-mRNA in cytoplasm is shown, and only nuclei are stained blue with DAPI. Comprehensive demonstration that the kit can specifically detect CTC from thyroid.
Example 3 evaluation of clinical diagnostic efficacy of kit
10mL of whole blood of 14 postoperative DTC patients and 12 healthy controls are collected, a filter membrane method is used for enriching CTC, a Diff (Diff-Quik) rapid staining method is used for preliminary detection of the DTC patients and the healthy controls, a Tg-IF (immunofluorescence) probe is used for detecting CTC in whole blood of 15 DTC postoperative patients and 6 healthy controls, the kit is used for detecting CTC in whole blood of 21 DTC patients and 15 normal healthy controls, and finally statistics and analysis are carried out on the results. As a result, it was found (fig. 3) that the number of CTCs in whole blood of the patient was 44.6 on average, whereas 8.2 CTCs were found on average in 5ml of whole blood of the healthy control, but there was no statistical difference between the two (P > 0.05) by statistical analysis, using Diff flash staining. Using Tg-IF probes, the number of CTCs in the patient group (average 3.5) was significantly higher than that in the normal control group (average 0.5) (P < 0.05); using the present kit, the number of CTCs in the patient group (average 23.5) was significantly higher than that in the normal control group (average 0.9) (P < 0.05). The results also show that the number of CTCs in the patient group detected by the Tg-mRNA FISH probe of the kit is obviously higher than that in the patient group detected by the Tg-IF probe method (P < 0.001); the quantity of the stained CTCs of the Diff-Quik is obviously higher than that of the CTCs detected by the kit, which shows that misdiagnosis is possibly caused by using the Diff-Quik staining, and misdiagnosis is easily caused by a Tg-IF probe method.
Diagnostic efficacy analysis showed (fig. 3) that the area under maximum curve (AUC) stained with Diff-Quik was 0.592 (95% ci:0.369-0.816, p=0.425), the cutoff value was 73.0CTCs, the sensitivity was 21.4%, and the specificity was 100.0%; the area under the maximum curve (AUC) using IF technique was 0.649 (95% ci: 0.471-0.827, p=0.132), the cutoff value was 1.5CTCs, the sensitivity was 42.9%, the specificity was 86.7%; the area under the maximum curve (AUC) when using the kit is 0.939 (95% CI:0.000-1.000, p < 0.001), the cutoff value is 4.5CTCs, the sensitivity is 73.3%, and the specificity is 100.0%. The result comparison shows that the kit has better diagnosis efficiency compared with the IF technology and the Diff-Quik.
The filter membrane method can effectively enrich CTC in whole blood of a patient, and the Diff rapid staining method can be used for identifying CTC of a DTC patient, but the false positive rate of the detection result is higher. Tg-IF probes can specifically detect CTC in DTC patients, but there is some false negative result. Compared with a Tg-IF probe and a Diff rapid staining method, the kit has good specificity, high accuracy and better diagnosis efficiency, can be used for detecting specific CTC of a DTC patient, and provides accurate thyroid patient prognosis and recurrence risk assessment basis for clinicians.
Sequence listing
<110> Jiangsu province people's hospital (first affiliated hospital of Nanjing medical university)
<120> differentiated thyroid cancer circulating tumor cell detection kit
<160> 48
<170> SIPOSequenceListing 1.0
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gattgaactg cgaggaaccg 20
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caatctgtca agcactgcat 20
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tttctggtca gaagtcatgc 20
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<400> 32
tggggttgta caatccagaa 20
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aaaggtgtct tgagttcctt 20
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tttctacatc ccatagactc 20
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<213> Artificial sequence (Artificial Sequence)
<400> 35
ttccattgac aatgacctgg 20
<210> 36
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 36
tccagtataa ctttcttccg 20
<210> 37
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 37
acggcaggcg agtgtaaaag 20
<210> 38
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 38
agaaatccaa agccagtgca 20
<210> 39
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 39
gctgttcaga gtgagacatg 20
<210> 40
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 40
gagacacttt ctctgtgagg 20
<210> 41
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 41
ttccgacaaa cagaggtctc 20
<210> 42
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 42
cttcacgaag cagaagtcgg 20
<210> 43
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 43
gaaggtacag atgcctcata 20
<210> 44
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 44
tggtgttgtg gaagaacacc 20
<210> 45
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 45
caagaaggag ccgtcgatag 20
<210> 46
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 46
catcattgag gacattggca 20
<210> 47
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 47
agaactccca atgagcagat 20
<210> 48
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 48
ttcaggagca tggtacatga 20

Claims (6)

1. A Tg high specificity mRNA nucleic acid probe composition is characterized by comprising 48 probes shown in SEQ ID NO.1-SEQ ID NO. 48.
2. Use of the Tg high specificity mRNA nucleic acid probe composition of claim 1 for preparing a differentiated thyroid cancer circulating tumor cell detection kit.
3. A kit for detecting differentiated thyroid cancer circulating tumor cells, comprising an 8 μm nanofiltration membrane and the Tg high specificity mRNA nucleic acid probe composition of claim 1.
4. The kit of claim 3, further comprising reagents related to solidification, hybridization of Tg probes, washing of the enriched cells.
5. A method for detecting thyroid cancer circulating tumor cells, which is not aimed at disease diagnosis, characterized in that circulating tumor cells in a blood sample are enriched and detected by using the kit of claim 3.
6. The method for detecting thyroid cancer circulating tumor cells according to claim 5, comprising the steps of:
(1) Enriching the circulating tumor cells in the blood sample using an 8 μm nanofiltration membrane;
(2) Solidifying the enriched cells, hybridizing and washing by using the Tg high specificity mRNA nucleic acid probe composition, and counting the number of CTCs in blood under a fluorescence microscope.
CN202210136153.3A 2022-02-15 2022-02-15 Differentiation type thyroid cancer circulating tumor cell detection kit Pending CN116640844A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202210136153.3A CN116640844A (en) 2022-02-15 2022-02-15 Differentiation type thyroid cancer circulating tumor cell detection kit

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202210136153.3A CN116640844A (en) 2022-02-15 2022-02-15 Differentiation type thyroid cancer circulating tumor cell detection kit

Publications (1)

Publication Number Publication Date
CN116640844A true CN116640844A (en) 2023-08-25

Family

ID=87638663

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202210136153.3A Pending CN116640844A (en) 2022-02-15 2022-02-15 Differentiation type thyroid cancer circulating tumor cell detection kit

Country Status (1)

Country Link
CN (1) CN116640844A (en)

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