CN107843731B - Kit for detecting pancreatic cancer cell markers in peripheral blood - Google Patents
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Abstract
The invention provides a kit for detecting pancreatic cancer cells in peripheral blood, which comprises magnetic beads combined with pancreatic cancer antibodies and corresponding buffer reagents, wherein the kit adopts molecular biology methods such as magnetic bead sorting and RT-PCR (reverse transcription-polymerase chain reaction) to detect the biological activity of tumor cells in blood circulation of pancreatic cancer patients, determines genes of drug treatment targets, determines the development and the prognosis of pancreatic cancer by detecting the expression levels of EpCAM, KRT19 and MUC16, and provides theoretical basis and experimental evidence for individualized treatment of pancreatic cancer. The kit provided by the invention has the advantages of small detection sample amount, small harm to patients, high detection sensitivity and detection rate of over 80%.
Description
Technical Field
The invention belongs to the technical field of biology, relates to a pancreatic cancer detection kit, and particularly relates to a kit for detecting a pancreatic cancer cell marker in peripheral blood with high accuracy.
Background
1.1 pancreatic cancer overview
Pancreatic cancer (pancreatic carcinoma) is a common malignancy of the digestive tract, with about 90% of ductal adenocarcinoma (PDAC) originating from the ductal epithelium, whose morbidity and mortality have increased significantly in recent years, with 90% of pancreatic cancer patients having a survival of no more than one year, less than 5% of more than five years, being one of the worst-case malignancies in advance. Factors influencing the unsatisfactory curative effect of pancreatic cancer include atypical early symptoms, complex pathophysiological mechanism, lack of early diagnosis and prognosis markers and the like. Although the diagnosis and treatment of tumors have been improved in recent years, the prognosis of many tumors has been improved, but the prognosis of pancreatic cancer patients has been least improved according to the statistics of the american cancer society. The high mortality rate of pancreatic cancer is due to: 1) although imaging methods play an important role in pancreatic cancer detection and treatment, it is still difficult to identify small lesions, pancreatitis, intraepithelial neoplasia and other benign lesions, from pancreatic cancer, with only 7% of pancreatic cancers being diagnosed at an early stage. 2) The lack of effective treatment for advanced pancreatic cancer results in poor prognosis in most patients, with a 5-year relative survival rate of 22% in patients with surgically resectable local lesions, and only 1-2% in patients with unresectable advanced metastatic lesions. 3) Pancreatic cancer has low survival rates due to its invasive biological phenotype, manifested by early local spread and metastasis.
From the economic point of view, the biological marker may become a more cost-effective early disease detection and diagnosis method, and the carbohydrate antigen CA199 clinically applied at present is used as the most classical marker of pancreatic cancer, and the sensitivity and specificity of the carbohydrate antigen CA199 are insufficient because the carbohydrate antigen is normal in the early stage of pancreatic cancer and is also increased in benign lesions such as pancreatitis or other tumors. In view of the fact that no single biological marker meets the requirement of initial diagnosis of pancreatic cancer, research and development of novel pancreatic cancer-related biological markers are urgently needed to distinguish healthy individuals from early pancreatic cancer or precursor lesions, so that the morbidity and mortality of diseases are reduced. Circulating Tumor Cells (CTCs) are tumor cells released from solid tumors or metastasis into peripheral blood circulation spontaneously or due to diagnosis and treatment operations, and can assist in early diagnosis and benign and malignant judgment, guide staging and molecular typing, assist in therapeutic drug screening and real-time monitoring of curative effect, prompt micrometastasis, prognosis and the like.
1.2 relevant factors affecting the prognosis of pancreatic cancer
For years, surgical treatment still remains the only effective means for radical treatment of pancreatic cancer, which has the leading position in pancreatic cancer treatment, and only radical resection of tumor tissue can lead patients to obtain longer survival time. While pancreatic cancer is insidious, 85% of patients are diagnosed at the late stage and miss the operation time. For unresectable pancreatic cancer, survival time after definitive diagnosis, even with adjuvant chemotherapy, is generally not longer than 6 months. Most research reports show that the prognosis of pancreatic cancer is not closely related to age, sex, family history of tumor, and tumor site. The association of diabetes with pancreatic cancer has been controversial, and studies have shown that a history of diabetes can increase the risk of pancreatic cancer, while diabetes can also reduce the post-operative survival rate of patients with pancreatic ductal adenocarcinoma. The prognosis of pancreatic cancer patients is related to peripancreatic invasion and distant metastasis, which are also the main bases of clinical staging of pancreatic cancer (pancreatic cancer staging is shown in Table 1: AJCC 8 th edition pancreatic cancer staging system). Early symptoms of pancreatic cancer are not obvious, and partial patients benefit from early discovery and are radically resected to obviously improve survival rate, so that early diagnosis of pancreatic cancer can effectively improve survival time of pancreatic cancer.
TABLE 1 AJCC 8 th edition pancreatic cancer staging System
1.3 clinical common pancreatic cancer detection technology
Currently, clinical detection of serological tumor markers for pancreatic cancer mainly includes CA199, CA242, CEA, MUC16, and the like. Among them, CA199 is one of the most commonly used markers for serological detection of pancreatic cancer, and CA199 is a mucin type glycoprotein tumor marker, which has an ascending trend in serum of patients with gastrointestinal tumor, and therefore, it is often used as a marker for gastrointestinal tumor and also a marker for the strongest pancreatic cancer sensitivity at present. It has also been shown that CA199 is positively correlated with the stage of cancer in patients, i.e., the expression level of CA199 increases with the progression of pancreatic cancer stages, and furthermore, the level is correlated with the prognosis of patients. However, the positive rate of the pancreatic cancer in the stage I is only 57.69%, and the finding of the pancreatic cancer in the early stage is not helpful greatly. However, since CA199 is also increased in obstructive jaundice, pancreatitis and various non-pancreatic tumors, and false positives are likely to occur, it cannot be used alone as a diagnostic standard for differentiating pancreatic cancer. MUC16 is a glycoprotein antigen expressed by coelomic epithelial cells in the development process of a carcinoembryonic, and is often used as a gynecological tumor marker such as ovarian cancer. Research shows that MUC16 in pancreatic cancer patients is also obviously higher than normal level, and meanwhile, in each stage, the expression level and the positive rate of MUC16 show an upward trend, and the MUC16 can be used as an independent prediction factor for poor prognosis of pancreatic ductal adenocarcinoma. However, these factors have their own defects and cannot be used alone as a diagnostic marker for pancreatic cancer. There is therefore a clinical need for an effective detection method that is less invasive, facilitates early diagnosis and can detect tumor progression in real time.
1.4 mechanism of pancreatic cancer metastasis
Since long, one has been plagued by the problem of "what determines which organ metastasizes", in 1889, Stephen Paget observed that breast cancer patients are more likely to develop liver metastases, which Paget thought to be unusual because other organs, such as the spleen, would likewise receive the same effect because the spleen and liver have the same blood flow, which prompted Paget to suggest a "seed-soil" theory that he hypothesized that certain tumor cells, i.e., seeds, would selectively clone to distant organs, i.e., soil, which provides an environment suitable for tumor cell growth "when a plant is initially sown, its seeds could be sown in any direction but they would only grow in suitable soil" so that certain organs must provide a suitable environment suitable for organ-specific metastasis resulting in organ selectivity. The process of metastasis of pancreatic cancer also supports the hypothesis that pancreatic cancer has a very low survival rate and is prone to distant metastasis, with liver metastasis being the most common. Hematogenous metastasis is the main way for pancreatic cancer metastasis, and tumor cells are shed from primary cancer tissues into blood, spread to distant tissues along with the blood flow and form metastases.
1.5 circulating tumor cells
Circulating tumor cells refer to tumor cells with primary tumors or metastatic foci falling into blood, and it was first proposed in 1869 that some of the CTCs entering the blood may have the ability to clone and grow to form metastases in a tissue-supported microenvironment, so that the number and molecular characteristics of CTCs can be used as a real-time noninvasive real-time "liquid biopsy" to provide clinical information about prognosis, treatment selection and effectiveness. The research of CTC is already a quite active field nowadays, a large number of researches indicate the existence and clinical significance of CTC in malignant tumor, these researches suggest that CTC is an independent prediction factor of poor prognosis, the progression-free survival and the total survival of patients with positive CTC detection are obviously shorter than those of patients with negative CTC detection, and the number of CTC is reduced after effective cytoreductive surgery or radiotherapy and chemotherapy, which suggests that CTC can assist in detecting the treatment effect so as to guide further treatment; in addition, the CTC also carries genetic information of primary tissues, and can provide a basis for individualized treatment through molecular level change.
In pancreatic ductal adenocarcinoma, high expression of EpCAM, KRT19, and MUC16, correlated with poor prognosis of pancreatic cancer.
In conclusion, the detection of the number of circulating tumor cells in the blood of pancreatic cancer patients has great theoretical and practical significance for the diagnosis and prognosis evaluation of pancreatic cancer.
Disclosure of Invention
The invention aims to provide a kit capable of accurately detecting a pancreatic cancer marker, mainly relates to a detection kit and a composite detection technology for collecting pancreatic cancer cells in peripheral blood of a pancreatic cancer patient, in particular to a detection technology and a kit for detecting pancreatic cancer cells (malignant tumor cells) with high accuracy, and particularly relates to a detection kit capable of detecting free pancreatic cancer cells remaining in blood after radiotherapy and chemotherapy treatment, wherein the detection result has important guiding significance for selection and prognosis of a pancreatic cancer treatment scheme.
The invention idea is as follows: the biological activity of tumor cells in blood circulation of pancreatic cancer patients is detected by molecular biological methods such as magnetic bead separation and RT-PCR (reverse transcription-polymerase chain reaction), the development and the prognosis of pancreatic cancer are determined by detecting the expression levels of EpCAM, KRT19 and MUC16, and theoretical basis and experimental evidence are provided for individualized treatment of pancreatic cancer.
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention provides a kit for detecting pancreatic cancer markers in peripheral blood, which comprises the following components:
magnetic beads with pancreatic cancer antibody bound:
magnetic beads to which oligonucleotide oligo (dT) is bound;
a buffer solution A; 100-
And (3) buffer solution B: 100-;
lysis/binding buffer 150-250 mL;
4.0-5.0mL of 10 Xbuffer solution;
dNTPs，5mM each 4.0-5.0mL
reverse transcriptase, 10000U/. mu.L 2.0-3.0mL
RNase inhibitor, 40U/. mu.L 0.4-0.6mL
2-3mL of 2X Hot Start PCR mixture
1-2mL of RNase-free water;
3.0-5.0mL of pancreatic cancer tumor marker primer 10 mu mol/L;
wherein the pancreatic cancer tumor marker is one or more of EpCAM, KRT19, MUC 16;
the EpCAM primer sequence is:
the upstream primer is shown as SEQ ID NO: 1, and the following components: 5'-TGAGCGAGTGAGAACCTA-3', respectively;
the downstream primer is shown as SEQ ID NO: 2, as shown in the figure: 5'-CACAACAATTCCAGCAAC-3', respectively;
the KRT19 primer sequence is as follows:
the upstream primer is shown as SEQ ID NO: 3, showing: 5'-CTGACACCATTCCTCCCT-3', respectively;
the downstream primer is shown as SEQ ID NO: 4, and (2) is as follows: 5'-CCGACGACTGGCGATA-3', respectively;
the MUC16 primer sequence is as follows:
the upstream primer is shown as SEQ ID NO: and 5, as follows: 5'-TCCCTGGATGCTGCTA-3', respectively;
the downstream primer is shown as SEQ ID NO: 6, showing: 5'-TGCTGAGGTGGCTATG-3', respectively;
the pancreatic cancer antibody is an anti-EpCAM monoclonal antibody and an anti-CK 20 monoclonal antibody.
Wherein the 2 Xhot start PCR mixture contains 5U/. mu.L of HotStarTaq DNA Polymerase.
Wherein the formula of the buffer solution A is as follows: 10mM Tris-HCl, pH 7.5; 0.15M LiCl; 1mM EDTA; 0.1% LiDS.
Wherein the formula of the buffer solution B is as follows: 10mM Tris-HCl, pH 7.5; 0.15M LiCl; 1mM EDTA.
Wherein the lysis/binding buffer formulation: 100mM Tris-HCl, pH 7.5; 500mM LiCl; 10mM EDTA, pH 8; 1% LiDS; 5mM dithioritol.
The tumor markers used for enriching the pancreatic cancer cells in the circulating blood are EpCAM and CK 20; while the markers that identify pancreatic cancer are EpCAM, KRT19, and MUC 16.
Preparing a primer:
the primers were each first diluted to 10. mu.M (i.e., 10. mu. mol/L), and then the primer mix was added with water to a concentration of 2. mu.M per primer and a final concentration of 0.2. mu.M per primer in the multiplex PCR reaction system.
In use, 25. mu.L of the antibody-labeled magnetic beads were used per 1mL of blood.
And (3) analyzing a reaction result:
analysis of PCR products using Agilent 2100 bioanalyzer:
the invention has the beneficial effects that:
the invention provides a kit for detecting a pancreatic cancer marker in peripheral blood, which has the advantages of small detection sample amount required by the kit, small damage to patients, high detection sensitivity, and low detection amount of cancer cells to 2 cell level, namely 2 free cancer cells in a sample can be detected, pancreatic cancer cells or pancreatic cancer stem cells can be determined and detected according to the content of the pancreatic cancer tumor marker, the detection rate can reach 80%, and theoretical basis and experimental evidence are provided for individualized treatment of recurrence and relapse of pancreatic cancer patients after treatment.
Drawings
FIG. 1 is a diagram of multiple PCR verification of the expression of each factor in the peripheral blood DNA of pancreatic ductal adenocarcinoma.
FIG. 2 is a dose-effect relationship diagram of EpCAM, KRT19 and Muc16 in the kit provided by the invention.
FIG. 3 shows the result of the expression of EpCAM, KRT19 and Muc16 in CTC of pancreatic cancer patients in the kit provided by the present invention.
Detailed Description
Reagents and apparatus used in the invention:
1. anti-EpCAM monoclonal antibody and anti-CK 20 monoclonal antibody were purchased from Abcom.
2. The magnetic beads used for binding antibody are selected from Invitrogen corporation
M-450Tosylactivated, the monoclonal antibody labeling amount is 500. mu.L of magnetic beads per 100. mu.g of antibody, labeling method and operation according to the instruction.
3. The magnetic beads to which oligo (dT) is bound are selected from Invitrogen, USA
Oligo(dT)25Magnetic beads.
4.10 × buffer: the reaction Buffer was a Sensiccript Reverse Transcriptase reaction Buffer (10 Xbuffer RT) from QIAGEN, USA.
5.2 × Hot Start PCR mix: the primer is a Multiplex PCR Master Mix of QIAGEN company, USA, wherein the HotStarTaq DNA Polymerase specification is the concentration: 5U/. mu.L.
Agilent 2100 bioanalyzer was purchased from Agilent technologies (China) Inc.
The reagents and instruments which are not indicated are all conventional articles for laboratories.
Example 1: preparation of magnetic beads to which pancreatic cancer antibodies were bound:
the antibodies used were anti-EpCAM monoclonal antibody and anti-CK 20 monoclonal antibody, and the 2 antibodies were labeled and then the 2 labeled magnetic beads were mixed and used.
The marking method comprises the following steps: magnetic beads manufactured by Invitrogen corporation of America were used
M-450 Tosylactivated. The marking method is carried out strictly according to the product specification.
The marking ratio is: each 100. mu.g of antibody was labeled with 500. mu.L of magnetic beads, and the labeled antibody was mixed in equal amounts.
Example 2: isolation of pancreatic cancer cells:
2.1 sample treatment:
5mL of peripheral blood of a pancreatic cancer patient is taken, EDTA is anticoagulated, and the pancreatic cancer patient is stored for 4 degrees and used within 48 hours.
2.2 magnetic bead treatment:
repeatedly washing magnetic beads (antibody-labeled magnetic beads, mixed magnetic beads for labeling two antibodies, and prepared antibody-labeled magnetic beads according to the proportion of adding 25 mu L of magnetic beads to 1mL of sample) combined with pancreatic cancer antibodies with PBS (phosphate buffer solution) for three times, separating the magnetic beads, and keeping on ice for later use;
the specific operation is as follows: sucking 125 mu L of marked magnetic beads, adding the magnetic beads into a centrifugal tube with the volume of 1.5mL, gently blowing and sucking the mixture by using a pipette, uniformly mixing the mixture (taking note that a vortex mixer cannot be used), placing the mixture on a magnetic bead enricher, standing the mixture for 1min to attach the magnetic beads to the wall of the centrifugal tube, and discarding the supernatant; then adding 1mL of PBS, placing on a magnetic bead enrichment device for 1min to attach the magnetic beads to the wall of the centrifugal tube, and removing the supernatant; washing with PBS for 3 times to remove antiseptic; after removing the supernatant, 200. mu.L of PBS was added and kept on ice.
And magnetic bead enrichment devices are used for separating magnetic beads in the subsequent steps.
2.3 pancreatic cancer cell sorting:
adding the sample and the antibody-labeled magnetic beads into a 15mL conical centrifuge tube, placing the reaction tube on a test tube rotary mixer at 4 ℃, incubating for 30min at the speed of 10rpm, separating the magnetic beads, and discarding the supernatant; the beads were washed with 1mL PBS, the supernatant removed to remove unwanted cells, and the beads were mixed with 1mL PBS and transferred to a new 1.5mL centrifuge tube for use.
2.4 pancreatic cancer cell lysis:
PBS was removed, 200. mu.L of lysis/binding buffer was added to the cells bound with the antibody-labeled magnetic beads and mixed, reacted in a water bath (or metal bath) at 55 ℃ for 5min to lyse the cells bound to the magnetic beads, mRNA was released into the supernatant, the centrifuge tube was placed on a magnet and allowed to stand for 5min, the supernatant was transferred to a new 1.5mL centrifuge tube, and the antibody-labeled magnetic beads were discarded.
Pancreatic cancer cell selection procedure was completed and sample mRNA was used for further experiments or mRNA was stored at-20 ℃ for up to 1 week with long term storage using-70 ℃.
Example 3: detection of pancreatic cancer cell markers:
3.1mRNA purification
Uniformly mixing 40 mu L of magnetic beads with oligonucleotide oligo (dT), adding the mixture into a 1.5mL centrifuge tube, adding 500 mu L of lysis/binding buffer solution for washing for 2 times, separating the magnetic beads, adding supernatant, uniformly mixing, and incubating at room temperature for 10min to enable mRNA to be bound with the magnetic beads; then separating the magnetic beads, washing the magnetic beads for 2 times by using 500 mu L of buffer solution A, separating the magnetic beads, washing the magnetic beads for 2 times by using 500 mu L of buffer solution B, separating the magnetic beads, washing the magnetic beads once by using 100 mu L of RNase-free water, separating the magnetic beads, re-suspending the magnetic beads by using 29.5 mu L of RNase-free water, incubating the magnetic beads in a water bath (or a metal bath) at 55 ℃ for 5min, and placing the magnetic beads on ice for 2min to obtain an mRNA/magnetic bead mixture.
The mRNA/magnetic bead mixture could not be stored and reverse transcription was performed immediately.
3.2 reverse transcription:
the reverse transcription step is as follows:
reverse transcription procedure:
storing at 37 deg.C for 60min, 93 deg.C for 5min, and 4 deg.C.
After completion of reverse transcription, PCR was continued or stored at-20 ℃ for up to 2 weeks.
The reaction was performed while RNase-free water was used instead of the sample as a negative control.
3.3PCR
The primer sequence is as follows:
the positive controls were: collecting pancreatic cancer cell line BxPC-3, extracting RNA, performing reverse transcription and translation to obtain positive control cDNA;
negative controls were: high purity water.
Reaction system
PCR procedure
15min at 95 ℃; 30s at 94 ℃, 90s at 58 ℃, 60s at 72 ℃ and 35 cycles; 30min at 60 ℃; infinity at 4 ℃
The PCR product was stored on ice or at-20 ℃.
3.5 analysis of results
The PCR products were analyzed by Agilent bioanalyzer for all experimental configurations.
1) All patient samples must have a band for the Actin gene (internal standard);
2) negative control for RT can not have a band greater than 80 nucleotides;
3) if the product is larger than 1kb, contamination by genomic DNA (introns) is indicated;
4) in the experiment, the result can be judged to be positive if any one of 3 positive strips in the PCR product is positive;
5) in the PCR result, 3 PCR products except Actin exist in the tumor cells, and the PCR product of EpCAM is related to the epithelial cells which are the sources of the tumor cells;
6) in the PCR result, other 3 bands than Actin are necessary to appear at each time, and can randomly appear. However, the lowest PCR detection should be 2 cells, i.e. any of 3 PCR products can be detected as long as 2 or more than 2 tumor cells or tumor stem cells are present in the sample to be detected.
First, the reliability of three identified markers was determined by multiplex PCR, and FIG. 1 shows the results of identifying three markers in BxPC-3 pancreatic cancer cell line. After RNA reverse transcription cDNA is extracted from the pancreatic cancer cell line, PCR results of EpCAM, KRT19 and MUC16 are respectively carried out, and Actin is selected as an internal reference. Secondly, as shown in fig. 2, fig. 2 shows the dose-effect relationship result of three markers, namely EpCAM, KRT19 and MUC16, by the sensitivity of the positive control analysis kit, it can be seen that when the number of cancer cells is 2 or more than 2, pancreatic cancer cells can be detected by using the kit provided by the present invention. And with the increase of the number of cancer cells, the amount of PCR products also increases, and the obvious dose-effect relationship is realized. The method has great application value for the prognosis evaluation of pancreatic cancer patients. FIG. 3 shows the sensitivity of three markers to peripheral blood samples of patients, BxPC-3 pancreatic cancer cell line is positive control, high-purity water is negative control, preoperative and postoperative peripheral blood of patients are selected, CTC is enriched according to the experimental process, then RNA is extracted, cDNA is subjected to reverse transcription, and then the results of the three markers are verified by multiplex PCR. The results show that free pancreatic cancer CTCs are detected in peripheral blood of patients before and after operation.
The embodiment shows that the kit for detecting pancreatic cancer cells in peripheral blood has simple and convenient detection method, small detection sample amount, small damage to patients and high detection sensitivity, 2 pancreatic cancer cells contained in the peripheral blood sample can be detected, the detection accuracy can reach 80%, and the detection result is credible.
The kit provided by the invention is mainly used for monitoring the pancreatic cancer patient after treatment, and provides reliable experimental data for doctors to evaluate whether the pancreatic cancer patient relapses after treatment and the treatment effect.
SEQUENCE LISTING
<110> Beijing cattle Gene technology Co., Ltd
Beijing university
Research institute for tumor prevention and treatment in Beijing
<120> kit for detecting pancreatic cancer cell marker in peripheral blood
<160> 8
<170> PatentIn version 3.3
<210> 1
<211> 18
<212> DNA
<213> Artificial sequence
<400> 1
tgagcgagtg agaaccta 18
<210> 2
<211> 18
<212> DNA
<213> Artificial sequence
<400> 2
cacaacaatt ccagcaac 18
<210> 3
<211> 18
<212> DNA
<213> Artificial sequence
<400> 3
ctgacaccat tcctccct 18
<210> 4
<211> 16
<212> DNA
<213> Artificial sequence
<400> 4
ccgacgactg gcgata 16
<210> 5
<211> 16
<212> DNA
<213> Artificial sequence
<400> 5
tccctggatg ctgcta 16
<210> 6
<211> 16
<212> DNA
<213> Artificial sequence
<400> 6
tgctgaggtg gctatg 16
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<211> 19
<212> DNA
<213> Artificial sequence
<400> 7
gaaatcgtgc gtgacatta 19
<210> 8
<211> 18
<212> DNA
<213> Artificial sequence
<400> 8
aggcagctcg tagctctt 18
Claims (4)
1. A kit for detecting pancreatic cancer cell markers in peripheral blood, which is characterized by comprising the following components:
magnetic beads with pancreatic cancer antibody bound:
magnetic beads to which oligonucleotide oligo (dT) is bound;
a buffer solution A; 100-
And (3) buffer solution B: 100-;
lysis/binding buffer 150-250 mL;
4.0-5.0mL of 10 Xbuffer solution;
dNTPs, 5mM each 4.0-5.0mL
Reverse transcriptase, 10000U/. mu.L 2.0-3.0mL
RNase inhibitor, 40U/. mu.L 0.4-0.6mL
2-3mL of 2X Hot Start PCR mixture
1-2mL of RNase-free water;
3.0-5.0mL of pancreatic cancer tumor marker primer 10 mu mol/L;
an internal standard ACTIN primer;
wherein the pancreatic cancer tumor markers are EpCAM, KRT19, and MUC 16;
the EpCAM primer sequence is:
the upstream primer is shown as SEQ ID NO: 1 is shown in the specification;
the downstream primer is shown as SEQ ID NO: 2 is shown in the specification;
the KRT19 primer sequence is as follows:
the upstream primer is shown as SEQ ID NO: 3 is shown in the specification;
the downstream primer is shown as SEQ ID NO: 4 is shown in the specification;
the MUC16 primer sequence is as follows:
the upstream primer is shown as SEQ ID NO: 5 is shown in the specification;
the downstream primer is shown as SEQ ID NO: 6 is shown in the specification; the internal standard ACTIN primer sequence is as follows:
the upstream primer is shown as SEQ ID NO: 7 is shown in the specification;
the downstream primer is shown as SEQ ID NO: 8 is shown in the specification; the pancreatic cancer antibody is an anti-EpCAM monoclonal antibody and an anti-CK 20 monoclonal antibody.
2. The kit of claim 1, wherein the 2 x hot start PCR mix comprises hot starttaq DNA Polymerase 5U/μ L.
3. The kit of claim 1, wherein buffer a is formulated as: 10mM Tris-HCl, pH 7.5; 0.15M LiCl; 1mM EDTA; 0.1% LiDS.
4. The kit of claim 1, wherein the buffer B is formulated as: 10mM Tris-HCl, pH 7.5; 0.15M LiCl; 1mM EDTA.
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| Multimarker gene analysis of circulating tumor cells in pancreatic cancer patients:a feasibility study;Andreia de Albuquerque，et al;《Oncology》;20120120;第82卷(第1期);摘要，第4页左栏第2段至第5页左栏第1段，表1，第9页左栏第2段至右栏第1段 * |
| Pathobiological Implications of MUC16 Expression in Pancreatic Cancer;Dhanya Haridas，et al;《PLoS ONE》;20111031;第6卷(第10期);摘要，第3页左栏第1-2段，倒数第1段，右栏倒数第1段 * |
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