CN114085907A - Method for determining pancreatic cancer progression by MSI2-Numb combined with metabolic molecules - Google Patents
Method for determining pancreatic cancer progression by MSI2-Numb combined with metabolic molecules Download PDFInfo
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Abstract
A method for determining pancreatic cancer progression by MSI2-Numb combined with metabolic molecules belongs to the technical field of detection, and comprises tumor tissue resection stage detection and blood prognosis stage detection. The tumor tissue resection stage detection comprises tumor tissue cell pretreatment, tumor tissue Musashi2 determination, tumor tissue Numb determination, blood metabolism molecule determination and judgment; the detection of the blood prognosis stage comprises the detection of blood Musashi2, the detection of blood Numb, the detection of blood metabolism molecules and prognosis; the invention provides a method for determining pancreatic cancer progression by combining MSI2-Numb with metabolic molecules, which can be used for screening pancreatic cancer progression by determining expression of Musashi2 and Numb in pancreatic cancer tissues and combining blood detection, can objectively evaluate clinical tumor progression, drug resistance and poor prognosis, is beneficial to drawing up an optimal treatment scheme, and has detection precision 20% higher than that of the existing detection method.
Description
Technical Field
The invention belongs to the technical field of medical instruments, and particularly relates to a method for determining pancreatic cancer progression by combining MSI2-Numb with metabolic molecules.
Background
Pancreatic cancer is a malignant tumor of the digestive tract which is extremely malignant and difficult to diagnose and treat, and about 90% of pancreatic cancers originate in ductal pancreatic cancer of the pancreatic duct epithelium and are one of the worst-prognosis malignant tumors. Early stage pancreatic cancer has low clinical diagnosis rate, high operative mortality rate and low cure rate. Pancreatic cancer is clinically characterized by the finding of advanced disease, short course of treatment, rapid disease progression and rapid deterioration. At present, the clinical lack of the early screening and diagnosis technology of pancreatic cancer results in the late stage pancreatic cancer discovery and the extremely limited diagnosis and treatment effect.
Among them, Musashi2(MSI2) and Numb play important roles in cell asymmetric division, maintenance of hematopoietic cell and neural cell line functions. There was a significant correlation between expression of Musashi2(MSI2) and Numb in pancreatic cancer tissue, which in combination objectively reflected the progression of the disease in pancreatic cancer patients and assessed the prognosis of pancreatic cancer patients.
In order to improve the accurate diagnosis and treatment level and cure rate of pancreatic cancer, it is important to determine the disease progression and prognosis of a pancreatic cancer patient.
Disclosure of Invention
Aiming at the lack of an accurate and effective pancreatic cancer tumor clinical progression screening and prognosis measuring method in the prior art, the invention provides a method for measuring pancreatic cancer progression by combining MSI2-Numb with metabolic molecules, which can screen the pancreatic cancer progression and evaluate the prognosis of a pancreatic cancer patient by measuring the expression of Musashi2(MSI2) and Numb in pancreatic cancer tissues and combining blood detection, thereby improving the detection precision and accuracy. The specific technical scheme is as follows:
a method for determining pancreatic cancer progression by MSI2-Numb binding to a metabolic molecule, comprising the steps of:
firstly, detecting the resection stage of tumor tissue:
step 1, tumor tissue pretreatment:
washing tumor tissue with DEPC water, and freezing at-80 deg.C in a freezing tube; placing 30-50 mg of tumor tissue into a 1.5ml centrifuge tube, adding 1.5ml Trizol, fully homogenizing, and standing at room temperature for 10 min; adding 300 μ l chloroform into each tube, mixing vigorously for 30sec, standing for 20min, centrifuging at 4 deg.C and 10000rpm for 15 min; gently sucking 400 μ l of the supernatant liquid into another new centrifuge tube, adding isopropanol with the same volume, gently inverting and mixing, and centrifuging at 4 ℃ and 10000rpm for 15 min; discarding the supernatant, adding 2ml of 75% alcohol to wash the precipitate, and centrifuging at 4 ℃ and 10000rpm for 15 min; removing the supernatant, airing at room temperature for 10min, adding 10 mu l of RNase-free water into each tube, and dissolving at 65 ℃ for 10-15 min;
step 2, determination of tumor tissue Musashi 2:
pretreating a tumor tissue, placing the tumor tissue in a PCR reaction system, adding a fluorescent dye Sypro and a hot start hTaq enzyme into the PCR reaction system, wherein the fluorescent dye Sypro can be specifically combined with Musashi2 to emit a fluorescent signal, so that the increase of the fluorescent signal and the increase of a PCR product are completely synchronous;
the working concentration of the fluorescent dye Sypro is 0.3 x-1 x, and the working concentration of the hot start hTaq enzyme is 0.03-0.1U/muL;
the PCR reaction system also comprises a PCR buffer solution, a fluorescence labeling Cy5, a dATP solution, a dGTP solution, a dCTP solution, a PCR protective agent, calcium ions and sterile double distilled water;
the PCR reaction system also comprises a forward primer of 8 mu mol/L, and the nucleotide sequence is as follows: AGGTTGAGCCATGCAGTCAT, respectively;
the PCR reaction system also comprises 8 mu mol/L reverse primer, and the nucleotide sequence is as follows: AGGTTGAGCCATGCAGTAGC, respectively;
the working concentration of the dATP solution is 50-80 mu mol/L; the working concentration of the dGTP solution is 50-90 mu mol/L; the working concentration of the dCTP solution is 60-80 mu mol/L; the working concentration of the fluorescent label Cy5 is 0.2X-0.8X;
the PCR protective agent is gelatin, Tween20 or dithiothreitol;
the Musashi2 template amount in the PCR reaction system is as follows: 5-10 ng of plasmid based on a 20 mu L reaction system, and 3-8 mmol/L of calcium ion concentration;
the quantitative PCR reaction conditions were: 4min at 90 ℃, 15s at 95 ℃, 35s at 50 ℃ and 40 cycles.
Step 3, determining tumor tissue Numb:
after tumor tissues are pretreated, the tumor tissues are placed in a PCR reaction system, a fluorescent dye Ruby and a hot start hTaq enzyme are added into the PCR reaction system, and the fluorescent dye Ruby can be specifically combined with Numb to emit a fluorescent signal, so that the increase of the fluorescent signal and the increase of a PCR product are completely synchronous;
the working concentration of the fluorescent dye Ruby is 0.5 x-1 x, and the working concentration of the hot start hTaq enzyme is 0.04-0.1U/muL;
the PCR reaction system also comprises a PCR buffer solution, a fluorescence labeling Cy3, a dUTP solution, a dGTP solution, a dCTP solution, a PCR protective agent, calcium ions and sterile double distilled water;
the PCR reaction system also comprises a forward primer of 10 mu mol/L, and the nucleotide sequence is as follows: GGCACCCAGCACAATGAAGA, respectively;
the PCR reaction system also comprises a reverse primer of 10 mu mol/L, and the nucleotide sequence is as follows: ACTCCTGCTTGCTGATCCAC, respectively;
the working concentration of the dUTP solution is 60-80 mu mol/L; the working concentration of the dGTP solution is 40-70 mu mol/L; the working concentration of the dCTP solution is 50-70 mu mol/L; the working concentration of the fluorescent label Cy3 is 0.4X-0.7X;
the PCR protective agent is gelatin, Tween20 or dithiothreitol;
the Numb template amount in the PCR reaction system is as follows: based on a 20 mu L reaction system, 6-12 ng of plasmid and 3-8 mmol/L of calcium ion concentration;
the quantitative PCR reaction conditions were: 3min at 94 ℃, 20s at 94 ℃ and 30s at 50 ℃ for 40 cycles.
Step 4, measuring blood metabolic molecules:
measuring the number of metabolic molecules with the molecular weight of 100-3000 daltons in the blood of a patient, and calculating the ratio of the number of the molecules with the signal-to-noise ratio higher than the optimal threshold value to the number of the detected metabolic molecules: mixing blood with nano silicon, sampling the mixed blood on a target plate of a mass spectrum, obtaining data (the precision is 0.001-0.1 dalton) by using a mass spectrometer, wherein the obtained data is the relative number value M of metabolic molecules with the length of more than 100 dalton, and then selecting the number value N of all metabolic molecules with the length of 100-3000 dalton and the signal-to-noise ratio of more than 2.10;
the mixing mass ratio of the blood to the nano silicon is 1: 0.6; the ratio of the number of molecules with signal-to-noise ratio higher than the optimal threshold value to the number of the selected detected metabolic molecules is calculated by adopting the following formula: the score ratio is the number of molecules with the signal-to-noise ratio higher than the optimal threshold value/the number of detected metabolic molecules, and the score ratio is N/M;
step 5, comprehensive judgment:
judging the degree of tumor deterioration according to the degree that the content of Musashi2 in the tumor tissue is higher than a normal value and the degree that the content of Numb in the tumor tissue is lower than the normal value, and in combination with the degree that the number of all detected metabolic molecules in plasma is 100-3000 daltons and the signal-to-noise ratio is higher than the normal value;
II, detecting a prognosis stage:
step 1, measuring blood Musashi 2:
pretreating blood of a postoperative patient, placing the blood into a PCR reaction system, adding a fluorescent dye Sypro and a hot start hTaq enzyme into the PCR reaction system, wherein the fluorescent dye Sypro can be specifically combined with Musashi2 to emit a fluorescent signal, so that the increase of the fluorescent signal and the increase of a PCR product are completely synchronous;
the working concentration of the fluorescent dye Sypro is 0.35 x-1 x, and the working concentration of the hot start hTaq enzyme is 0.04-0.1U/muL;
the PCR reaction system also comprises a PCR buffer solution, a fluorescence labeling Cy5, a dATP solution, a dGTP solution, a dCTP solution, a PCR protective agent, calcium ions and sterile double distilled water;
the PCR reaction system also comprises a forward primer of 9 mu mol/L, and the nucleotide sequence is as follows: AGGTTGAGCCATGCAGTCAT, respectively;
the PCR reaction system also comprises a 9 mu mol/L reverse primer, and the nucleotide sequence is as follows: AGGTTGAGCCATGCAGTAGC, respectively;
the working concentration of the dATP solution is 90-95 mu mol/L; the working concentration of the dGTP solution is 100-120 mu mol/L; the working concentration of the dCTP solution is 100-120 mu mol/L; the working concentration of the fluorescent label Cy5 is 0.6X-0.8X;
the PCR protective agent is gelatin, Tween20 or dithiothreitol;
the Musashi2 template amount in the PCR reaction system is as follows: 5-10 ng of plasmid based on a 20 mu L reaction system, and 6-8 mmol/L of calcium ion concentration;
the quantitative PCR reaction conditions were: 4min at 90 ℃, 15s at 95 ℃, 35s at 50 ℃ and 40 cycles.
Step 2, measuring blood Numb:
pretreating blood of a postoperative patient, placing the blood into a PCR reaction system, adding a fluorescent dye Ruby and a hot start hTaq enzyme into the PCR reaction system, wherein the fluorescent dye Ruby can be specifically combined with Numb to emit a fluorescent signal, so that the increase of the fluorescent signal and the increase of a PCR product are completely synchronous;
the working concentration of the fluorescent dye Ruby is 0.8 x-1 x, and the working concentration of the hot start hTaq enzyme is 0.06-0.1U/muL;
the PCR reaction system also comprises a PCR buffer solution, a fluorescence labeling Cy3, a dUTP solution, a dGTP solution, a dCTP solution, a PCR protective agent, calcium ions and sterile double distilled water;
the PCR reaction system also comprises a forward primer of 12 mu mol/L, and the nucleotide sequence is as follows: GGCACCCAGCACAATGAAGA, respectively;
the PCR reaction system also comprises a reverse primer of 12 mu mol/L, and the nucleotide sequence is as follows: ACTCCTGCTTGCTGATCCAC, respectively;
the working concentration of the dUTP solution is 100-120 mu mol/L; the working concentration of the dGTP solution is 80-100 mu mol/L; the working concentration of the dCTP solution is 80-100 mu mol/L; the working concentration of the fluorescent label Cy3 is 0.5X-0.8X;
the PCR protective agent is gelatin, Tween20 or dithiothreitol;
the Numb template amount in the PCR reaction system is as follows: based on a 20 mu L reaction system, 6-12 ng of plasmid and 5-10 mmol/L of calcium ion concentration;
the quantitative PCR reaction conditions were: 3min at 94 ℃, 20s at 94 ℃ and 30s at 50 ℃ for 40 cycles.
Step 3, measuring blood metabolic molecules:
measuring the number of metabolic molecules with the molecular weight of 100-3000 daltons in the blood of a patient, and calculating the ratio of the number of the molecules with the signal-to-noise ratio higher than the optimal threshold value to the number of the detected metabolic molecules: mixing blood with nano silicon, sampling the mixed blood on a target plate of a mass spectrum, obtaining data (the precision is 0.001-0.1 dalton) by using a mass spectrometer, wherein the obtained data is the relative number value M of metabolic molecules with the length of more than 100 dalton, and then selecting the number value N of all metabolic molecules with the length of 100-3000 dalton and the signal-to-noise ratio of more than 2.10;
the mixing mass ratio of the blood to the nano silicon is 1: 0.6; the ratio of the number of molecules with signal-to-noise ratio higher than the optimal threshold value to the number of the selected detected metabolic molecules is calculated by adopting the following formula: the score ratio is the number of molecules with the signal-to-noise ratio higher than the optimal threshold value/the number of detected metabolic molecules, and the score ratio is N/M;
step 4, prognosis judgment:
performing multiple blood detections in a prognosis stage, and comparing the detection result of each time with the detection result of the last time;
determining that the content of Musashi2 in the blood of the postoperative patient is higher than the content of Musashi2 in the blood of the last measurement, the content of Numb in the blood of the postoperative patient is lower than the content of Numb in the blood of the last measurement, and determining that all detected molecular weights in the blood of the postoperative patient are 100-3000 daltons, and the ratio of the number of metabolic molecules with the signal-to-noise ratio of more than 2.10 is higher than the last measurement, thus indicating that the disease condition of the patient develops or further worsens; otherwise, the patient is indicated to have improved condition.
Compared with the prior art, the method for determining pancreatic cancer progression by combining MSI2-Numb with metabolic molecules has the beneficial effects that:
the method comprehensively measures the disease progress by combining the level of Musashi2(MSI2) and Numb in pancreatic cancer tissues with the number of blood metabolic molecules, and the evaluation of the pancreatic tumor deterioration degree is more accurate.
Secondly, the invention determines the expression of Musashi2 and Numb in the blood of a patient with prognosis, and comprehensively determines the postoperative disease progression by combining blood metabolic molecules: determining that the content of Musashi2 in the blood of the postoperative patient is higher than the content of Musashi2 in the blood of the last measurement, the content of Numb in the blood of the postoperative patient is lower than the content of Numb in the blood of the last measurement, and determining that all detected molecular weights in the blood of the postoperative patient are 100-3000 daltons, and the ratio of the number of metabolic molecules with the signal-to-noise ratio of more than 2.10 is higher than the last measurement, thus indicating that the disease condition of the patient develops or further worsens; otherwise, the patient is indicated to have improved condition.
And thirdly, the comprehensive determination of the three indexes is more accurate, wherein the MSI2-Numb depends on a p53 pathway to regulate the proliferation and the drug resistance of pancreatic cancer cells. The determination method can objectively evaluate clinical progress, drug resistance and poor prognosis of the tumor, is beneficial to drawing up an optimal treatment scheme, and can improve the detection precision by 20 percent compared with the existing metabolic molecule detection method which only depends on.
Fourthly, the Musashi2 quantitative PCR method provided by the invention uses hot start hTaq enzyme, has proper calcium ion concentration, can well inhibit nonspecific amplification, avoids false positive and high background results, and can obtain a single peak on a dissolution curve, so that the fluorescent dye Sypro has a better effect and high sensitivity.
The Numb quantitative PCR method disclosed by the invention adopts proper calcium ion concentration, so that nonspecific amplification can be well inhibited, false positive and high background results are avoided, a single peak can be obtained by a dissolution curve, the effect of the fluorescent dye Ruby is better exerted, the sensitivity is high, and the prognosis of a patient is influenced by detecting low-expression Numb.
In conclusion, the specific Musashi2 and Numb quantitative PCR method provided by the invention has more accurate detection; and the pancreatic cancer condition is detected through the expression difference of Musashi2 and Numb and the content of metabolic molecules, so that the prognosis is more accurate.
Drawings
FIG. 1 is a flowchart of the detection of tumor tissue resection stage according to the present invention;
FIG. 2 is a flow chart of the detection of the prognostic stage according to the present invention.
Detailed Description
The invention will be further described with reference to the following examples and figures 1-2, but the invention is not limited to these examples.
Example 1
A method for determining pancreatic cancer progression by MSI2-Numb binding to a metabolic molecule, comprising the steps of:
firstly, detecting the tumor tissue resection stage, as shown in fig. 1:
step 1, tumor tissue pretreatment:
washing tumor tissue with DEPC water, and freezing at-80 deg.C in a freezing tube; placing 30mg tumor tissue in a 1.5ml centrifuge tube, adding 1.5ml Trizol, homogenizing thoroughly, and standing at room temperature for 10 min; adding 300 μ l chloroform into each tube, mixing vigorously for 30sec, standing for 20min, centrifuging at 4 deg.C and 10000rpm for 15 min; gently sucking 400 μ l of the supernatant liquid into another new centrifuge tube, adding isopropanol with the same volume, gently inverting and mixing, and centrifuging at 4 ℃ and 10000rpm for 15 min; discarding the supernatant, adding 2ml of 75% alcohol to wash the precipitate, and centrifuging at 4 ℃ and 10000rpm for 15 min; discarding supernatant, air drying at room temperature for 10min, adding 10 μ l of RNase-free water into each tube, and dissolving at 65 deg.C for 10 min;
step 2, determination of tumor tissue Musashi 2:
pretreating a tumor tissue, placing the tumor tissue in a PCR reaction system, adding a fluorescent dye Sypro and a hot start hTaq enzyme into the PCR reaction system, wherein the fluorescent dye Sypro can be specifically combined with Musashi2 to emit a fluorescent signal, so that the increase of the fluorescent signal and the increase of a PCR product are completely synchronous;
the working concentration of the fluorescent dye Sypro is 0.3 x, and the working concentration of the hot start hTaq enzyme is 0.03U/mu L;
the PCR reaction system also comprises a PCR buffer solution, a fluorescence labeling Cy5, a dATP solution, a dGTP solution, a dCTP solution, a PCR protective agent, calcium ions and sterile double distilled water;
the PCR reaction system also comprises a forward primer of 8 mu mol/L, and the nucleotide sequence is as follows: AGGTTGAGCCATGCAGTCAT, respectively;
the PCR reaction system also comprises 8 mu mol/L reverse primer, and the nucleotide sequence is as follows: AGGTTGAGCCATGCAGTAGC, respectively;
the working concentration of the dATP solution is 50 mu mol/L; the working concentration of the dGTP solution is 50 mu mol/L; the working concentration of the dCTP solution is 60 mu mol/L; the working concentration of the fluorescently labeled Cy5 was 0.2 ×;
the PCR protective agent is gelatin, Tween20 or dithiothreitol;
the Musashi2 template amount in the PCR reaction system is as follows: calculated by a 20 mu L reaction system, 5ng of plasmid and 3mmol/L of calcium ion concentration;
the quantitative PCR reaction conditions were: 4min at 90 ℃, 15s at 95 ℃, 35s at 50 ℃ and 40 cycles.
Step 3, determining tumor tissue Numb:
after tumor tissues are pretreated, the tumor tissues are placed in a PCR reaction system, a fluorescent dye Ruby and a hot start hTaq enzyme are added into the PCR reaction system, and the fluorescent dye Ruby can be specifically combined with Numb to emit a fluorescent signal, so that the increase of the fluorescent signal and the increase of a PCR product are completely synchronous;
the working concentration of the fluorescent dye Ruby is 0.5 x, and the working concentration of the hot start hTaq enzyme is 0.04U/muL;
the PCR reaction system also comprises a PCR buffer solution, a fluorescence labeling Cy3, a dUTP solution, a dGTP solution, a dCTP solution, a PCR protective agent, calcium ions and sterile double distilled water;
the PCR reaction system also comprises a forward primer of 10 mu mol/L, and the nucleotide sequence is as follows: GGCACCCAGCACAATGAAGA, respectively;
the PCR reaction system also comprises a reverse primer of 10 mu mol/L, and the nucleotide sequence is as follows: ACTCCTGCTTGCTGATCCAC, respectively;
the working concentration of the dUTP solution is 60 mu mol/L; the working concentration of the dGTP solution is 40 mu mol/L; the working concentration of the dCTP solution is 50 mu mol/L; the working concentration of the fluorescently labeled Cy3 was 0.4 ×;
the PCR protective agent is gelatin, Tween20 or dithiothreitol;
the Numb template amount in the PCR reaction system is as follows: based on a 20 mu L reaction system, the plasmid is 6ng, and the calcium ion concentration is 3 mmol/L;
the quantitative PCR reaction conditions were: 3min at 94 ℃, 20s at 94 ℃ and 30s at 50 ℃ for 40 cycles.
Step 4, measuring blood metabolic molecules:
measuring the number of metabolic molecules with the molecular weight of 100-3000 daltons in the blood of a patient, and calculating the ratio of the number of the molecules with the signal-to-noise ratio higher than the optimal threshold value to the number of the detected metabolic molecules: mixing blood with nano silicon, sampling the mixed blood on a target plate of a mass spectrum, obtaining data (the precision is 0.001-0.1 dalton) by using a mass spectrometer, wherein the obtained data is the relative number value M of metabolic molecules with the length of more than 100 dalton, and then selecting the number value N of all metabolic molecules with the length of 100-3000 dalton and the signal-to-noise ratio of more than 2.10;
the mixing mass ratio of the blood to the nano silicon is 1: 0.6; the ratio of the number of molecules with signal-to-noise ratio higher than the optimal threshold value to the number of the selected detected metabolic molecules is calculated by adopting the following formula: the score ratio is the number of molecules with the signal-to-noise ratio higher than the optimal threshold value/the number of detected metabolic molecules, and the score ratio is N/M;
step 5, comprehensive judgment:
judging the degree of tumor deterioration according to the degree that the content of Musashi2 in the tumor tissue is higher than a normal value and the degree that the content of Numb in the tumor tissue is lower than the normal value, and in combination with the degree that the number of all detected metabolic molecules in plasma is 100-3000 daltons and the signal-to-noise ratio is higher than the normal value;
secondly, detection of the prognosis stage, as shown in fig. 2:
step 1, measuring blood Musashi 2:
pretreating blood of a postoperative patient, placing the blood into a PCR reaction system, adding a fluorescent dye Sypro and a hot start hTaq enzyme into the PCR reaction system, wherein the fluorescent dye Sypro can be specifically combined with Musashi2 to emit a fluorescent signal, so that the increase of the fluorescent signal and the increase of a PCR product are completely synchronous;
the working concentration of the fluorescent dye Sypro is 0.35 x-1 x, and the working concentration of the hot start hTaq enzyme is 0.04-0.1U/muL;
the PCR reaction system also comprises a PCR buffer solution, a fluorescence labeling Cy5, a dATP solution, a dGTP solution, a dCTP solution, a PCR protective agent, calcium ions and sterile double distilled water;
the PCR reaction system also comprises a forward primer of 9 mu mol/L, and the nucleotide sequence is as follows: AGGTTGAGCCATGCAGTCAT, respectively;
the PCR reaction system also comprises a 9 mu mol/L reverse primer, and the nucleotide sequence is as follows: AGGTTGAGCCATGCAGTAGC, respectively;
the working concentration of the dATP solution is 90-95 mu mol/L; the working concentration of the dGTP solution is 100-120 mu mol/L; the working concentration of the dCTP solution is 100-120 mu mol/L; the working concentration of the fluorescent label Cy5 is 0.6X-0.8X;
the PCR protective agent is gelatin, Tween20 or dithiothreitol;
the Musashi2 template amount in the PCR reaction system is as follows: 5-10 ng of plasmid based on a 20 mu L reaction system, and 6-8 mmol/L of calcium ion concentration;
the quantitative PCR reaction conditions were: 4min at 90 ℃, 15s at 95 ℃, 35s at 50 ℃ and 40 cycles.
Step 2, measuring blood Numb:
pretreating blood of a postoperative patient, placing the blood into a PCR reaction system, adding a fluorescent dye Ruby and a hot start hTaq enzyme into the PCR reaction system, wherein the fluorescent dye Ruby can be specifically combined with Numb to emit a fluorescent signal, so that the increase of the fluorescent signal and the increase of a PCR product are completely synchronous;
the working concentration of the fluorescent dye Ruby is 0.8 x-1 x, and the working concentration of the hot start hTaq enzyme is 0.06-0.1U/muL;
the PCR reaction system also comprises a PCR buffer solution, a fluorescence labeling Cy3, a dUTP solution, a dGTP solution, a dCTP solution, a PCR protective agent, calcium ions and sterile double distilled water;
the PCR reaction system also comprises a forward primer of 12 mu mol/L, and the nucleotide sequence is as follows: GGCACCCAGCACAATGAAGA, respectively;
the PCR reaction system also comprises a reverse primer of 12 mu mol/L, and the nucleotide sequence is as follows: ACTCCTGCTTGCTGATCCAC, respectively;
the working concentration of the dUTP solution is 100-120 mu mol/L; the working concentration of the dGTP solution is 80-100 mu mol/L; the working concentration of the dCTP solution is 80-100 mu mol/L; the working concentration of the fluorescent label Cy3 is 0.5X-0.8X;
the PCR protective agent is gelatin, Tween20 or dithiothreitol;
the Numb template amount in the PCR reaction system is as follows: based on a 20 mu L reaction system, 6-12 ng of plasmid and 5-10 mmol/L of calcium ion concentration;
the quantitative PCR reaction conditions were: 3min at 94 ℃, 20s at 94 ℃ and 30s at 50 ℃ for 40 cycles.
Step 3, measuring blood metabolic molecules:
measuring the number of metabolic molecules with the molecular weight of 100-3000 daltons in the blood of a patient, and calculating the ratio of the number of the molecules with the signal-to-noise ratio higher than the optimal threshold value to the number of the detected metabolic molecules: mixing blood with nano silicon, sampling the mixed blood on a target plate of a mass spectrum, obtaining data (the precision is 0.001-0.1 dalton) by using a mass spectrometer, wherein the obtained data is the relative number value M of metabolic molecules with the length of more than 100 dalton, and then selecting the number value N of all metabolic molecules with the length of 100-3000 dalton and the signal-to-noise ratio of more than 2.10;
the mixing mass ratio of the blood to the nano silicon is 1: 0.6; the ratio of the number of molecules with signal-to-noise ratio higher than the optimal threshold value to the number of the selected detected metabolic molecules is calculated by adopting the following formula: the score ratio is the number of molecules with the signal-to-noise ratio higher than the optimal threshold value/the number of detected metabolic molecules, and the score ratio is N/M;
step 4, prognosis judgment:
performing multiple blood detections in a prognosis stage, and comparing the detection result of each time with the detection result of the last time;
determining that the content of Musashi2 in the blood of the postoperative patient is higher than the content of Musashi2 in the blood of the last measurement, the content of Numb in the blood of the postoperative patient is lower than the content of Numb in the blood of the last measurement, and determining that all detected molecular weights in the blood of the postoperative patient are 100-3000 daltons, and the ratio of the number of metabolic molecules with the signal-to-noise ratio of more than 2.10 is higher than the last measurement, thus indicating that the disease condition of the patient develops or further worsens; otherwise, the patient is indicated to have improved condition.
The fluorescent dyes Sypro and Ruby used in this example were purchased from Higashi Biotech, Inc; the hot start hTaq enzyme was purchased from New England Biolabs (NEB); PCR buffer, dATP solution, dUTP solution, dGTP solution and dCTP solution were purchased from Kyoto Biotech, Inc., Shanghai; the forward primer, the reverse primer, the PCR protectant and the like are purchased from Higashi Biotech Co., Ltd.
In this example, a pancreatic cancer patient is evaluated, and the tumor deterioration degree is judged by detecting the content of Musashi2 and Numb in tumor tissue cells and the metabolic molecular data in blood at one time of resection operation. And then, measuring Musashi2, Numb and metabolic molecule data in the blood of the patient after one operation at intervals of 1 month, carrying out prognosis, measuring the content of Musashi2 in the blood for the second time to be higher than the content of Musashi2 in the blood for the first time, measuring the content of the Numb in the blood to be lower than the content of the Numb in the blood for the first time, and measuring the proportion of all detected molecular weights in blood plasma of 100-3000 daltons and the number of metabolic molecules with the signal-to-noise ratio of more than 2.10 to be higher than that of the first measured value, thereby indicating that the condition of the patient is further worsened and changing other treatment schemes.
Example 2
A method for determining pancreatic cancer progression by MSI2-Numb binding to a metabolic molecule, comprising the steps of:
firstly, detecting the tumor tissue resection stage, as shown in fig. 1:
step 1, tumor tissue pretreatment:
washing tumor tissue with DEPC water, and freezing at-80 deg.C in a freezing tube; placing 35mg tumor tissue in a 1.5ml centrifuge tube, adding 1.5ml Trizol, homogenizing thoroughly, and standing at room temperature for 10 min; adding 300 μ l chloroform into each tube, mixing vigorously for 30sec, standing for 20min, centrifuging at 4 deg.C and 10000rpm for 15 min; gently sucking 400 μ l of the supernatant liquid into another new centrifuge tube, adding isopropanol with the same volume, gently inverting and mixing, and centrifuging at 4 ℃ and 10000rpm for 15 min; discarding the supernatant, adding 2ml of 75% alcohol to wash the precipitate, and centrifuging at 4 ℃ and 10000rpm for 15 min; discarding supernatant, air drying at room temperature for 10min, adding 10 μ l of RNase-free water into each tube, and dissolving at 65 deg.C for 12 min;
step 2, determination of tumor tissue Musashi 2:
pretreating a tumor tissue, placing the tumor tissue in a PCR reaction system, adding a fluorescent dye Sypro and a hot start hTaq enzyme into the PCR reaction system, wherein the fluorescent dye Sypro can be specifically combined with Musashi2 to emit a fluorescent signal, so that the increase of the fluorescent signal and the increase of a PCR product are completely synchronous;
the working concentration of the fluorescent dye Sypro is 0.5 x, and the working concentration of the hot start hTaq enzyme is 0.06U/muL;
the PCR reaction system also comprises a PCR buffer solution, a fluorescence labeling Cy5, a dATP solution, a dGTP solution, a dCTP solution, a PCR protective agent, calcium ions and sterile double distilled water;
the PCR reaction system also comprises a forward primer of 8 mu mol/L, and the nucleotide sequence is as follows: AGGTTGAGCCATGCAGTCAT, respectively;
the PCR reaction system also comprises 8 mu mol/L reverse primer, and the nucleotide sequence is as follows: AGGTTGAGCCATGCAGTAGC, respectively;
the working concentration of the dATP solution is 60 mu mol/L; the working concentration of the dGTP solution is 60 mu mol/L; the working concentration of the dCTP solution is 70 mu mol/L; the working concentration of the fluorescently labeled Cy5 was 0.3 ×;
the PCR protective agent is gelatin, Tween20 or dithiothreitol;
the Musashi2 template amount in the PCR reaction system is as follows: calculated by a 20 mu L reaction system, the plasmid is 6ng, and the calcium ion concentration is 4 mmol/L;
the quantitative PCR reaction conditions were: 4min at 90 ℃, 15s at 95 ℃, 35s at 50 ℃ and 40 cycles.
Step 3, determining tumor tissue Numb:
after tumor tissues are pretreated, the tumor tissues are placed in a PCR reaction system, a fluorescent dye Ruby and a hot start hTaq enzyme are added into the PCR reaction system, and the fluorescent dye Ruby can be specifically combined with Numb to emit a fluorescent signal, so that the increase of the fluorescent signal and the increase of a PCR product are completely synchronous;
the working concentration of the fluorescent dye Ruby is 0.6 x, and the working concentration of the hot start hTaq enzyme is 0.05U/muL;
the PCR reaction system also comprises a PCR buffer solution, a fluorescence labeling Cy3, a dUTP solution, a dGTP solution, a dCTP solution, a PCR protective agent, calcium ions and sterile double distilled water;
the PCR reaction system also comprises a forward primer of 10 mu mol/L, and the nucleotide sequence is as follows: GGCACCCAGCACAATGAAGA, respectively;
the PCR reaction system also comprises a reverse primer of 10 mu mol/L, and the nucleotide sequence is as follows: ACTCCTGCTTGCTGATCCAC, respectively;
the working concentration of the dUTP solution is 65 mu mol/L; the working concentration of the dGTP solution is 45 mu mol/L; the working concentration of the dCTP solution is 50 mu mol/L; the working concentration of the fluorescently labeled Cy3 was 0.5 ×;
the PCR protective agent is gelatin, Tween20 or dithiothreitol;
the Numb template amount in the PCR reaction system is as follows: calculated by a 20 mu L reaction system, the plasmid is 8ng, and the calcium ion concentration is 5 mmol/L;
the quantitative PCR reaction conditions were: 3min at 94 ℃, 20s at 94 ℃ and 30s at 50 ℃ for 40 cycles.
Step 4, measuring blood metabolic molecules:
measuring the number of metabolic molecules with the molecular weight of 100-3000 daltons in the blood of a patient, and calculating the ratio of the number of the molecules with the signal-to-noise ratio higher than the optimal threshold value to the number of the detected metabolic molecules: mixing blood with nano silicon, sampling the mixed blood on a target plate of a mass spectrum, obtaining data (the precision is 0.001-0.1 dalton) by using a mass spectrometer, wherein the obtained data is the relative number value M of metabolic molecules with the length of more than 100 dalton, and then selecting the number value N of all metabolic molecules with the length of 100-3000 dalton and the signal-to-noise ratio of more than 2.10;
the mixing mass ratio of the blood to the nano silicon is 1: 0.6; the ratio of the number of molecules with signal-to-noise ratio higher than the optimal threshold value to the number of the selected detected metabolic molecules is calculated by adopting the following formula: the score ratio is the number of molecules with the signal-to-noise ratio higher than the optimal threshold value/the number of detected metabolic molecules, and the score ratio is N/M;
step 5, comprehensive judgment:
judging the degree of tumor deterioration according to the degree that the content of Musashi2 in the tumor tissue is higher than a normal value and the degree that the content of Numb in the tumor tissue is lower than the normal value, and in combination with the degree that the number of all detected metabolic molecules in plasma is 100-3000 daltons and the signal-to-noise ratio is higher than the normal value;
secondly, detection of the prognosis stage, as shown in fig. 2:
step 1, measuring blood Musashi 2:
pretreating blood of a postoperative patient, placing the blood into a PCR reaction system, adding a fluorescent dye Sypro and a hot start hTaq enzyme into the PCR reaction system, wherein the fluorescent dye Sypro can be specifically combined with Musashi2 to emit a fluorescent signal, so that the increase of the fluorescent signal and the increase of a PCR product are completely synchronous;
the working concentration of the fluorescent dye Sypro is 0.6 x, and the working concentration of the hot start hTaq enzyme is 0.06U/muL;
the PCR reaction system also comprises a PCR buffer solution, a fluorescence labeling Cy5, a dATP solution, a dGTP solution, a dCTP solution, a PCR protective agent, calcium ions and sterile double distilled water;
the PCR reaction system also comprises a forward primer of 9 mu mol/L, and the nucleotide sequence is as follows: AGGTTGAGCCATGCAGTCAT, respectively;
the PCR reaction system also comprises a 9 mu mol/L reverse primer, and the nucleotide sequence is as follows: AGGTTGAGCCATGCAGTAGC, respectively;
the working concentration of the dATP solution is 92 mu mol/L; the working concentration of the dGTP solution is 110 mu mol/L; the working concentration of the dCTP solution is 110 mu mol/L; the working concentration of the fluorescently labeled Cy5 was 0.6 ×;
the PCR protective agent is gelatin, Tween20 or dithiothreitol;
the Musashi2 template amount in the PCR reaction system is as follows: calculated by a 20 mu L reaction system, the plasmid is 8ng, and the calcium ion concentration is 6 mmol/L;
the quantitative PCR reaction conditions were: 4min at 90 ℃, 15s at 95 ℃, 35s at 50 ℃ and 40 cycles.
Step 2, measuring blood Numb:
pretreating blood of a postoperative patient, placing the blood into a PCR reaction system, adding a fluorescent dye Ruby and a hot start hTaq enzyme into the PCR reaction system, wherein the fluorescent dye Ruby can be specifically combined with Numb to emit a fluorescent signal, so that the increase of the fluorescent signal and the increase of a PCR product are completely synchronous;
the working concentration of the fluorescent dye Ruby is 0.8 x, and the working concentration of the hot start hTaq enzyme is 0.08U/muL;
the PCR reaction system also comprises a PCR buffer solution, a fluorescence labeling Cy3, a dUTP solution, a dGTP solution, a dCTP solution, a PCR protective agent, calcium ions and sterile double distilled water;
the PCR reaction system also comprises a forward primer of 12 mu mol/L, and the nucleotide sequence is as follows: GGCACCCAGCACAATGAAGA, respectively;
the PCR reaction system also comprises a reverse primer of 12 mu mol/L, and the nucleotide sequence is as follows: ACTCCTGCTTGCTGATCCAC, respectively;
the working concentration of the dUTP solution is 110 mu mol/L; the working concentration of the dGTP solution is 90 mu mol/L; the working concentration of the dCTP solution is 90 mu mol/L; the working concentration of the fluorescently labeled Cy3 was 0.6 ×;
the PCR protective agent is gelatin, Tween20 or dithiothreitol;
the Numb template amount in the PCR reaction system is as follows: calculated by a 20 mu L reaction system, the plasmid is 8ng, and the calcium ion concentration is 8 mmol/L;
the quantitative PCR reaction conditions were: 3min at 94 ℃, 20s at 94 ℃ and 30s at 50 ℃ for 40 cycles.
Step 3, measuring blood metabolic molecules:
measuring the number of metabolic molecules with the molecular weight of 100-3000 daltons in the blood of a patient, and calculating the ratio of the number of the molecules with the signal-to-noise ratio higher than the optimal threshold value to the number of the detected metabolic molecules: mixing blood with nano silicon, sampling the mixed blood on a target plate of a mass spectrum, obtaining data (the precision is 0.001-0.1 dalton) by using a mass spectrometer, wherein the obtained data is the relative number value M of metabolic molecules with the length of more than 100 dalton, and then selecting the number value N of all metabolic molecules with the length of 100-3000 dalton and the signal-to-noise ratio of more than 2.10;
the mixing mass ratio of the blood to the nano silicon is 1: 0.6; the ratio of the number of molecules with signal-to-noise ratio higher than the optimal threshold value to the number of the selected detected metabolic molecules is calculated by adopting the following formula: the score ratio is the number of molecules with the signal-to-noise ratio higher than the optimal threshold value/the number of detected metabolic molecules, and the score ratio is N/M;
step 4, prognosis judgment:
performing multiple blood detections in a prognosis stage, and comparing the detection result of each time with the detection result of the last time;
determining that the content of Musashi2 in the blood of the postoperative patient is higher than the content of Musashi2 in the blood of the last measurement, the content of Numb in the blood of the postoperative patient is lower than the content of Numb in the blood of the last measurement, and determining that all detected molecular weights in the blood of the postoperative patient are 100-3000 daltons, and the ratio of the number of metabolic molecules with the signal-to-noise ratio of more than 2.10 is higher than the last measurement, thus indicating that the disease condition of the patient develops or further worsens; otherwise, the patient is indicated to have improved condition.
The fluorescent dyes Sypro and Ruby used in this example were purchased from Higashi Biotech, Inc; the hot start hTaq enzyme was purchased from New England Biolabs (NEB); PCR buffer, dATP solution, dUTP solution, dGTP solution and dCTP solution were purchased from Kyoto Biotech, Inc., Shanghai; the forward primer, the reverse primer, the PCR protectant and the like are purchased from Higashi Biotech Co., Ltd.
In this example, a pancreatic cancer patient is evaluated, and the tumor deterioration degree is judged by detecting the content of Musashi2 and Numb in tumor tissue cells and the metabolic molecular data in blood at one time of resection operation. And then, measuring Musashi2, Numb and metabolic molecule data in the blood of the patient after one operation at intervals of half a month for prognosis, measuring that the content of Musashi2 in the blood is lower than that of Musashi2 in the blood measured for the first time, measuring that the content of the Numb in the blood is higher than that of the Numb in the blood measured for the first time, and measuring that the proportion of all detected molecular weights in plasma is 100-3000 daltons and the number of metabolic molecules with the signal-to-noise ratio of more than 2.10 is lower than that of the first measured value, thereby indicating that the illness state of the patient is improved and continuing to observe.
Example 3
A method for determining pancreatic cancer progression by MSI2-Numb binding to a metabolic molecule, comprising the steps of:
firstly, detecting the tumor tissue resection stage, as shown in fig. 1:
step 1, tumor tissue pretreatment:
washing tumor tissue with DEPC water, and freezing at-80 deg.C in a freezing tube; putting 45mg of tumor tissue into a 1.5ml centrifuge tube, adding 1.5ml Trizol, homogenizing fully, and standing for 10min at room temperature; adding 300 μ l chloroform into each tube, mixing vigorously for 30sec, standing for 20min, centrifuging at 4 deg.C and 10000rpm for 15 min; gently sucking 400 μ l of the supernatant liquid into another new centrifuge tube, adding isopropanol with the same volume, gently inverting and mixing, and centrifuging at 4 ℃ and 10000rpm for 15 min; discarding the supernatant, adding 2ml of 75% alcohol to wash the precipitate, and centrifuging at 4 ℃ and 10000rpm for 15 min; discarding supernatant, air drying at room temperature for 10min, adding 10 μ l of RNase-free water into each tube, and dissolving at 65 deg.C for 15 min;
step 2, determination of tumor tissue Musashi 2:
pretreating a tumor tissue, placing the tumor tissue in a PCR reaction system, adding a fluorescent dye Sypro and a hot start hTaq enzyme into the PCR reaction system, wherein the fluorescent dye Sypro can be specifically combined with Musashi2 to emit a fluorescent signal, so that the increase of the fluorescent signal and the increase of a PCR product are completely synchronous;
the working concentration of the fluorescent dye Sypro is 0.8 x, and the working concentration of the hot start hTaq enzyme is 0.08U/muL;
the PCR reaction system also comprises a PCR buffer solution, a fluorescence labeling Cy5, a dATP solution, a dGTP solution, a dCTP solution, a PCR protective agent, calcium ions and sterile double distilled water;
the PCR reaction system also comprises a forward primer of 8 mu mol/L, and the nucleotide sequence is as follows: AGGTTGAGCCATGCAGTCAT, respectively;
the PCR reaction system also comprises 8 mu mol/L reverse primer, and the nucleotide sequence is as follows: AGGTTGAGCCATGCAGTAGC, respectively;
the working concentration of the dATP solution is 75 mu mol/L; the working concentration of the dGTP solution is 80 mu mol/L; the working concentration of the dCTP solution is 75 mu mol/L; the working concentration of the fluorescently labeled Cy5 was 0.7 ×;
the PCR protective agent is gelatin, Tween20 or dithiothreitol;
the Musashi2 template amount in the PCR reaction system is as follows: calculated by a 20 mu L reaction system, the plasmid is 7ng, and the calcium ion concentration is 6 mmol/L;
the quantitative PCR reaction conditions were: 4min at 90 ℃, 15s at 95 ℃, 35s at 50 ℃ and 40 cycles.
Step 3, determining tumor tissue Numb:
after tumor tissues are pretreated, the tumor tissues are placed in a PCR reaction system, a fluorescent dye Ruby and a hot start hTaq enzyme are added into the PCR reaction system, and the fluorescent dye Ruby can be specifically combined with Numb to emit a fluorescent signal, so that the increase of the fluorescent signal and the increase of a PCR product are completely synchronous;
the working concentration of the fluorescent dye Ruby is 0.9 x, and the working concentration of the hot start hTaq enzyme is 0.07U/muL;
the PCR reaction system also comprises a PCR buffer solution, a fluorescence labeling Cy3, a dUTP solution, a dGTP solution, a dCTP solution, a PCR protective agent, calcium ions and sterile double distilled water;
the PCR reaction system also comprises a forward primer of 10 mu mol/L, and the nucleotide sequence is as follows: GGCACCCAGCACAATGAAGA, respectively;
the PCR reaction system also comprises a reverse primer of 10 mu mol/L, and the nucleotide sequence is as follows: ACTCCTGCTTGCTGATCCAC, respectively;
the working concentration of the dUTP solution is 75 mu mol/L; the working concentration of the dGTP solution is 45 mu mol/L; the working concentration of the dCTP solution is 65 mu mol/L; the working concentration of the fluorescently labeled Cy3 was 0.55 ×;
the PCR protective agent is gelatin, Tween20 or dithiothreitol;
the Numb template amount in the PCR reaction system is as follows: calculated by a 20 mu L reaction system, the plasmid is 7ng, and the calcium ion concentration is 6 mmol/L;
the quantitative PCR reaction conditions were: 3min at 94 ℃, 20s at 94 ℃ and 30s at 50 ℃ for 40 cycles.
Step 4, measuring blood metabolic molecules:
measuring the number of metabolic molecules with the molecular weight of 100-3000 daltons in the blood of a patient, and calculating the ratio of the number of the molecules with the signal-to-noise ratio higher than the optimal threshold value to the number of the detected metabolic molecules: mixing blood with nano silicon, sampling the mixed blood on a target plate of a mass spectrum, obtaining data (the precision is 0.001-0.1 dalton) by using a mass spectrometer, wherein the obtained data is the relative number value M of metabolic molecules with the length of more than 100 dalton, and then selecting the number value N of all metabolic molecules with the length of 100-3000 dalton and the signal-to-noise ratio of more than 2.10;
the mixing mass ratio of the blood to the nano silicon is 1: 0.6; the ratio of the number of molecules with signal-to-noise ratio higher than the optimal threshold value to the number of the selected detected metabolic molecules is calculated by adopting the following formula: the score ratio is the number of molecules with the signal-to-noise ratio higher than the optimal threshold value/the number of detected metabolic molecules, and the score ratio is N/M;
step 5, comprehensive judgment:
judging the degree of tumor deterioration according to the degree that the content of Musashi2 in the tumor tissue is higher than a normal value and the degree that the content of Numb in the tumor tissue is lower than the normal value, and in combination with the degree that the number of all detected metabolic molecules in plasma is 100-3000 daltons and the signal-to-noise ratio is higher than the normal value;
secondly, detection of the prognosis stage, as shown in fig. 2:
step 1, measuring blood Musashi 2:
pretreating blood of a postoperative patient, placing the blood into a PCR reaction system, adding a fluorescent dye Sypro and a hot start hTaq enzyme into the PCR reaction system, wherein the fluorescent dye Sypro can be specifically combined with Musashi2 to emit a fluorescent signal, so that the increase of the fluorescent signal and the increase of a PCR product are completely synchronous;
the working concentration of the fluorescent dye Sypro is 0.35 x, and the working concentration of the hot start hTaq enzyme is 0.04U/muL;
the PCR reaction system also comprises a PCR buffer solution, a fluorescence labeling Cy5, a dATP solution, a dGTP solution, a dCTP solution, a PCR protective agent, calcium ions and sterile double distilled water;
the PCR reaction system also comprises a forward primer of 9 mu mol/L, and the nucleotide sequence is as follows: AGGTTGAGCCATGCAGTCAT, respectively;
the PCR reaction system also comprises a 9 mu mol/L reverse primer, and the nucleotide sequence is as follows: AGGTTGAGCCATGCAGTAGC, respectively;
the working concentration of the dATP solution is 90 mu mol/L; the working concentration of the dGTP solution is 100 mu mol/L; the working concentration of the dCTP solution is 100 mu mol/L; the working concentration of the fluorescently labeled Cy5 was 0.6 ×;
the PCR protective agent is gelatin, Tween20 or dithiothreitol;
the Musashi2 template amount in the PCR reaction system is as follows: calculated by a 20 mu L reaction system, the plasmid is 5ng, and the calcium ion concentration is 6 mmol/L;
the quantitative PCR reaction conditions were: 4min at 90 ℃, 15s at 95 ℃, 35s at 50 ℃ and 40 cycles.
Step 2, measuring blood Numb:
pretreating blood of a postoperative patient, placing the blood into a PCR reaction system, adding a fluorescent dye Ruby and a hot start hTaq enzyme into the PCR reaction system, wherein the fluorescent dye Ruby can be specifically combined with Numb to emit a fluorescent signal, so that the increase of the fluorescent signal and the increase of a PCR product are completely synchronous;
the working concentration of the fluorescent dye Ruby is 0.8 x, and the working concentration of the hot start hTaq enzyme is 0.06U/muL;
the PCR reaction system also comprises a PCR buffer solution, a fluorescence labeling Cy3, a dUTP solution, a dGTP solution, a dCTP solution, a PCR protective agent, calcium ions and sterile double distilled water;
the PCR reaction system also comprises a forward primer of 12 mu mol/L, and the nucleotide sequence is as follows: GGCACCCAGCACAATGAAGA, respectively;
the PCR reaction system also comprises a reverse primer of 12 mu mol/L, and the nucleotide sequence is as follows: ACTCCTGCTTGCTGATCCAC, respectively;
the working concentration of the dUTP solution is 100 mu mol/L; the working concentration of the dGTP solution is 80 mu mol/L; the working concentration of the dCTP solution is 80 mu mol/L; the working concentration of the fluorescently labeled Cy3 was 0.5 ×;
the PCR protective agent is gelatin, Tween20 or dithiothreitol;
the Numb template amount in the PCR reaction system is as follows: based on a 20 mu L reaction system, the plasmid is 6ng, and the calcium ion concentration is 5 mmol/L;
the quantitative PCR reaction conditions were: 3min at 94 ℃, 20s at 94 ℃ and 30s at 50 ℃ for 40 cycles.
Step 3, measuring blood metabolic molecules:
measuring the number of metabolic molecules with the molecular weight of 100-3000 daltons in the blood of a patient, and calculating the ratio of the number of the molecules with the signal-to-noise ratio higher than the optimal threshold value to the number of the detected metabolic molecules: mixing blood with nano silicon, sampling the mixed blood on a target plate of a mass spectrum, obtaining data (the precision is 0.001-0.1 dalton) by using a mass spectrometer, wherein the obtained data is the relative number value M of metabolic molecules with the length of more than 100 dalton, and then selecting the number value N of all metabolic molecules with the length of 100-3000 dalton and the signal-to-noise ratio of more than 2.10;
the mixing mass ratio of the blood to the nano silicon is 1: 0.6; the ratio of the number of molecules with signal-to-noise ratio higher than the optimal threshold value to the number of the selected detected metabolic molecules is calculated by adopting the following formula: the score ratio is the number of molecules with the signal-to-noise ratio higher than the optimal threshold value/the number of detected metabolic molecules, and the score ratio is N/M;
step 4, prognosis judgment:
performing multiple blood detections in a prognosis stage, and comparing the detection result of each time with the detection result of the last time;
determining that the content of Musashi2 in the blood of the postoperative patient is higher than the content of Musashi2 in the blood of the last measurement, the content of Numb in the blood of the postoperative patient is lower than the content of Numb in the blood of the last measurement, and determining that all detected molecular weights in the blood of the postoperative patient are 100-3000 daltons, and the ratio of the number of metabolic molecules with the signal-to-noise ratio of more than 2.10 is higher than the last measurement, thus indicating that the disease condition of the patient develops or further worsens; otherwise, the patient is indicated to have improved condition.
The fluorescent dyes Sypro and Ruby used in this example were purchased from Higashi Biotech, Inc; the hot start hTaq enzyme was purchased from New England Biolabs (NEB); PCR buffer, dATP solution, dUTP solution, dGTP solution and dCTP solution were purchased from Kyoto Biotech, Inc., Shanghai; the forward primer, the reverse primer, the PCR protectant and the like are purchased from Higashi Biotech Co., Ltd.
In this example, a pancreatic cancer patient is evaluated, and the tumor deterioration degree is judged by detecting the content of Musashi2 and Numb in tumor tissue cells and the metabolic molecular data in blood at one time of resection operation. And then, measuring Musashi2, Numb and metabolic molecule data in the blood of the patient after one operation at intervals of 1 month, carrying out prognosis, measuring the content of Musashi2 in the blood for the second time to be higher than the content of Musashi2 in the blood for the first time, measuring the content of the Numb in the blood to be lower than the content of the Numb in the blood for the first time, and measuring the proportion of all detected molecular weights in blood plasma of 100-3000 daltons and the number of metabolic molecules with the signal-to-noise ratio of more than 2.10 to be higher than the first measured value, thereby indicating that the condition of the patient is further worsened and changing other treatment schemes.
Example 4
A method for determining pancreatic cancer progression by MSI2-Numb binding to a metabolic molecule, comprising the steps of:
firstly, detecting the tumor tissue resection stage, as shown in fig. 1:
step 1, tumor tissue pretreatment:
washing tumor tissue with DEPC water, and freezing at-80 deg.C in a freezing tube; placing 50mg tumor tissue in a 1.5ml centrifuge tube, adding 1.5ml Trizol, homogenizing thoroughly, and standing at room temperature for 10 min; adding 300 μ l chloroform into each tube, mixing vigorously for 30sec, standing for 20min, centrifuging at 4 deg.C and 10000rpm for 15 min; gently sucking 400 μ l of the supernatant liquid into another new centrifuge tube, adding isopropanol with the same volume, gently inverting and mixing, and centrifuging at 4 ℃ and 10000rpm for 15 min; discarding the supernatant, adding 2ml of 75% alcohol to wash the precipitate, and centrifuging at 4 ℃ and 10000rpm for 15 min; discarding supernatant, air drying at room temperature for 10min, adding 10 μ l of RNase-free water into each tube, and dissolving at 65 deg.C for 15 min;
step 2, determination of tumor tissue Musashi 2:
pretreating a tumor tissue, placing the tumor tissue in a PCR reaction system, adding a fluorescent dye Sypro and a hot start hTaq enzyme into the PCR reaction system, wherein the fluorescent dye Sypro can be specifically combined with Musashi2 to emit a fluorescent signal, so that the increase of the fluorescent signal and the increase of a PCR product are completely synchronous;
the working concentration of the fluorescent dye Sypro is 1 x, and the working concentration of the hot start hTaq enzyme is 0.1U/muL;
the PCR reaction system also comprises a PCR buffer solution, a fluorescence labeling Cy5, a dATP solution, a dGTP solution, a dCTP solution, a PCR protective agent, calcium ions and sterile double distilled water;
the PCR reaction system also comprises a forward primer of 8 mu mol/L, and the nucleotide sequence is as follows: AGGTTGAGCCATGCAGTCAT, respectively;
the PCR reaction system also comprises 8 mu mol/L reverse primer, and the nucleotide sequence is as follows: AGGTTGAGCCATGCAGTAGC, respectively;
the working concentration of the dATP solution is 80 mu mol/L; the working concentration of the dGTP solution is 90 mu mol/L; the working concentration of the dCTP solution is 80 mu mol/L; the working concentration of the fluorescently labeled Cy5 was 0.8 ×;
the PCR protective agent is gelatin, Tween20 or dithiothreitol;
the Musashi2 template amount in the PCR reaction system is as follows: calculated by a 20 mu L reaction system, the plasmid is 10ng, and the calcium ion concentration is 8 mmol/L;
the quantitative PCR reaction conditions were: 4min at 90 ℃, 15s at 95 ℃, 35s at 50 ℃ and 40 cycles.
Step 3, determining tumor tissue Numb:
after tumor tissues are pretreated, the tumor tissues are placed in a PCR reaction system, a fluorescent dye Ruby and a hot start hTaq enzyme are added into the PCR reaction system, and the fluorescent dye Ruby can be specifically combined with Numb to emit a fluorescent signal, so that the increase of the fluorescent signal and the increase of a PCR product are completely synchronous;
the working concentration of the fluorescent dye Ruby is 1 x, and the working concentration of the hot start hTaq enzyme is 0.1U/muL;
the PCR reaction system also comprises a PCR buffer solution, a fluorescence labeling Cy3, a dUTP solution, a dGTP solution, a dCTP solution, a PCR protective agent, calcium ions and sterile double distilled water;
the PCR reaction system also comprises a forward primer of 10 mu mol/L, and the nucleotide sequence is as follows: GGCACCCAGCACAATGAAGA, respectively;
the PCR reaction system also comprises a reverse primer of 10 mu mol/L, and the nucleotide sequence is as follows: ACTCCTGCTTGCTGATCCAC, respectively;
the working concentration of the dUTP solution is 80 mu mol/L; the working concentration of the dGTP solution is 70 mu mol/L; the working concentration of the dCTP solution is 70 mu mol/L; the working concentration of the fluorescently labeled Cy3 was 0.7 ×;
the PCR protective agent is gelatin, Tween20 or dithiothreitol;
the Numb template amount in the PCR reaction system is as follows: calculated by a 20 mu L reaction system, the plasmid is 12ng, and the calcium ion concentration is 8 mmol/L;
the quantitative PCR reaction conditions were: 3min at 94 ℃, 20s at 94 ℃ and 30s at 50 ℃ for 40 cycles.
Step 4, measuring blood metabolic molecules:
measuring the number of metabolic molecules with the molecular weight of 100-3000 daltons in the blood of a patient, and calculating the ratio of the number of the molecules with the signal-to-noise ratio higher than the optimal threshold value to the number of the detected metabolic molecules: mixing blood with nano silicon, sampling the mixed blood on a target plate of a mass spectrum, obtaining data (the precision is 0.001-0.1 dalton) by using a mass spectrometer, wherein the obtained data is the relative number value M of metabolic molecules with the length of more than 100 dalton, and then selecting the number value N of all metabolic molecules with the length of 100-3000 dalton and the signal-to-noise ratio of more than 2.10;
the mixing mass ratio of the blood to the nano silicon is 1: 0.6; the ratio of the number of molecules with signal-to-noise ratio higher than the optimal threshold value to the number of the selected detected metabolic molecules is calculated by adopting the following formula: the score ratio is the number of molecules with the signal-to-noise ratio higher than the optimal threshold value/the number of detected metabolic molecules, and the score ratio is N/M;
step 5, comprehensive judgment:
judging the degree of tumor deterioration according to the degree that the content of Musashi2 in the tumor tissue is higher than a normal value and the degree that the content of Numb in the tumor tissue is lower than the normal value, and in combination with the degree that the number of all detected metabolic molecules in plasma is 100-3000 daltons and the signal-to-noise ratio is higher than the normal value;
secondly, detection of the prognosis stage, as shown in fig. 2:
step 1, measuring blood Musashi 2:
pretreating blood of a postoperative patient, placing the blood into a PCR reaction system, adding a fluorescent dye Sypro and a hot start hTaq enzyme into the PCR reaction system, wherein the fluorescent dye Sypro can be specifically combined with Musashi2 to emit a fluorescent signal, so that the increase of the fluorescent signal and the increase of a PCR product are completely synchronous;
the working concentration of the fluorescent dye Sypro is 1 x, and the working concentration of the hot start hTaq enzyme is 0.1U/muL;
the PCR reaction system also comprises a PCR buffer solution, a fluorescence labeling Cy5, a dATP solution, a dGTP solution, a dCTP solution, a PCR protective agent, calcium ions and sterile double distilled water;
the PCR reaction system also comprises a forward primer of 9 mu mol/L, and the nucleotide sequence is as follows: AGGTTGAGCCATGCAGTCAT, respectively;
the PCR reaction system also comprises a 9 mu mol/L reverse primer, and the nucleotide sequence is as follows: AGGTTGAGCCATGCAGTAGC, respectively;
the working concentration of the dATP solution is 95 mu mol/L; the working concentration of the dGTP solution is 120 mu mol/L; the working concentration of the dCTP solution is 120 mu mol/L; the working concentration of the fluorescently labeled Cy5 was 0.8 ×;
the PCR protective agent is gelatin, Tween20 or dithiothreitol;
the Musashi2 template amount in the PCR reaction system is as follows: calculated by a 20 mu L reaction system, the plasmid is 10ng, and the calcium ion concentration is 8 mmol/L;
the quantitative PCR reaction conditions were: 4min at 90 ℃, 15s at 95 ℃, 35s at 50 ℃ and 40 cycles.
Step 2, measuring blood Numb:
pretreating blood of a postoperative patient, placing the blood into a PCR reaction system, adding a fluorescent dye Ruby and a hot start hTaq enzyme into the PCR reaction system, wherein the fluorescent dye Ruby can be specifically combined with Numb to emit a fluorescent signal, so that the increase of the fluorescent signal and the increase of a PCR product are completely synchronous;
the working concentration of the fluorescent dye Ruby is 1 x, and the working concentration of the hot start hTaq enzyme is 0.1U/muL;
the PCR reaction system also comprises a PCR buffer solution, a fluorescence labeling Cy3, a dUTP solution, a dGTP solution, a dCTP solution, a PCR protective agent, calcium ions and sterile double distilled water;
the PCR reaction system also comprises a forward primer of 12 mu mol/L, and the nucleotide sequence is as follows: GGCACCCAGCACAATGAAGA, respectively;
the PCR reaction system also comprises a reverse primer of 12 mu mol/L, and the nucleotide sequence is as follows: ACTCCTGCTTGCTGATCCAC, respectively;
the working concentration of the dUTP solution is 120 mu mol/L; the working concentration of the dGTP solution is 100 mu mol/L; the working concentration of the dCTP solution is 100 mu mol/L; the working concentration of the fluorescently labeled Cy3 was 0.8 ×;
the PCR protective agent is gelatin, Tween20 or dithiothreitol;
the Numb template amount in the PCR reaction system is as follows: calculated by a 20 mu L reaction system, the plasmid is 12ng, and the calcium ion concentration is 10 mmol/L;
the quantitative PCR reaction conditions were: 3min at 94 ℃, 20s at 94 ℃ and 30s at 50 ℃ for 40 cycles.
Step 3, measuring blood metabolic molecules:
measuring the number of metabolic molecules with the molecular weight of 100-3000 daltons in the blood of a patient, and calculating the ratio of the number of the molecules with the signal-to-noise ratio higher than the optimal threshold value to the number of the detected metabolic molecules: mixing blood with nano silicon, sampling the mixed blood on a target plate of a mass spectrum, obtaining data (the precision is 0.001-0.1 dalton) by using a mass spectrometer, wherein the obtained data is the relative number value M of metabolic molecules with the length of more than 100 dalton, and then selecting the number value N of all metabolic molecules with the length of 100-3000 dalton and the signal-to-noise ratio of more than 2.10;
the mixing mass ratio of the blood to the nano silicon is 1: 0.6; the ratio of the number of molecules with signal-to-noise ratio higher than the optimal threshold value to the number of the selected detected metabolic molecules is calculated by adopting the following formula: the score ratio is the number of molecules with the signal-to-noise ratio higher than the optimal threshold value/the number of detected metabolic molecules, and the score ratio is N/M;
step 4, prognosis judgment:
performing multiple blood detections in a prognosis stage, and comparing the detection result of each time with the detection result of the last time;
determining that the content of Musashi2 in the blood of the postoperative patient is higher than the content of Musashi2 in the blood of the last measurement, the content of Numb in the blood of the postoperative patient is lower than the content of Numb in the blood of the last measurement, and determining that all detected molecular weights in the blood of the postoperative patient are 100-3000 daltons, and the ratio of the number of metabolic molecules with the signal-to-noise ratio of more than 2.10 is higher than the last measurement, thus indicating that the disease condition of the patient develops or further worsens; otherwise, the patient is indicated to have improved condition.
The fluorescent dyes Sypro and Ruby used in this example were purchased from Higashi Biotech, Inc; the hot start hTaq enzyme was purchased from New England Biolabs (NEB); PCR buffer, dATP solution, dUTP solution, dGTP solution and dCTP solution were purchased from Kyoto Biotech, Inc., Shanghai; the forward primer, the reverse primer, the PCR protectant and the like are purchased from Higashi Biotech Co., Ltd.
In this example, a pancreatic cancer patient is evaluated, and the tumor deterioration degree is judged by detecting the content of Musashi2 and Numb in tumor tissue cells and the metabolic molecular data in blood at one time of resection operation. And then, measuring Musashi2, Numb and metabolic molecule data in the blood of the operation patient at intervals of half a month for prognosis, measuring the Musashi2 content in the blood for the second time to be lower than the Musashi2 content in the blood for the first time, measuring the Numb content in the blood to be higher than the Numb content in the blood for the first time, and measuring the proportion of all detected molecular weights in plasma to be 100-3000 daltons and the number of metabolic molecules with the signal-to-noise ratio of more than 2.10 to be lower than the first measured value, thereby indicating that the condition of the patient is improved.
Claims (8)
- A method for determining pancreatic cancer progression by MSI2-Numb binding to a metabolic molecule, comprising the steps of:firstly, detecting the tumor tissue resection stage:step 1, tumor tissue pretreatment:washing tumor tissue with DEPC water, and freezing at-80 deg.C in a freezing tube; placing 30-50 mg of tumor tissue into a 1.5ml centrifuge tube, adding 1.5ml Trizol, fully homogenizing, and standing at room temperature for 10 min; adding 300 μ l chloroform into each tube, mixing vigorously for 30sec, standing for 20min, centrifuging at 4 deg.C and 10000rpm for 15 min; gently sucking 400 μ l of the supernatant liquid into another new centrifuge tube, adding isopropanol with the same volume, gently inverting and mixing, and centrifuging at 4 ℃ and 10000rpm for 15 min; discarding the supernatant, adding 2ml of 75% alcohol to wash the precipitate, and centrifuging at 4 ℃ and 10000rpm for 15 min; removing the supernatant, airing at room temperature for 10min, adding 10 mu l of RNase-free water into each tube, and dissolving at 65 ℃ for 10-15 min;step 2, determination of tumor tissue Musashi 2:pretreating a tumor tissue, placing the tumor tissue in a PCR reaction system, adding a fluorescent dye Sypro and a hot start hTaq enzyme into the PCR reaction system, wherein the fluorescent dye Sypro can be specifically combined with Musashi2 to emit a fluorescent signal, so that the increase of the fluorescent signal and the increase of a PCR product are completely synchronous;the PCR reaction system also comprises a PCR buffer solution, a fluorescence labeling Cy5, a dATP solution, a dGTP solution, a dCTP solution, a PCR protective agent, calcium ions and sterile double distilled water;the PCR reaction system also comprises a forward primer of 8 mu mol/L, and the nucleotide sequence is as follows: AGGTTGAGCCATGCAGTCAT, respectively;the PCR reaction system also comprises 8 mu mol/L reverse primer, and the nucleotide sequence is as follows: AGGTTGAGCCATGCAGTAGC, respectively;the Musashi2 template amount in the PCR reaction system is as follows: 5-10 ng of plasmid based on a 20 mu L reaction system, and 3-8 mmol/L of calcium ion concentration;the quantitative PCR reaction conditions were: 4min at 90 ℃, 15s at 95 ℃, 35s at 50 ℃ and 40 cycles.Step 3, determining tumor tissue Numb:after tumor tissues are pretreated, the tumor tissues are placed in a PCR reaction system, a fluorescent dye Ruby and a hot start hTaq enzyme are added into the PCR reaction system, and the fluorescent dye Ruby can be specifically combined with Numb to emit a fluorescent signal, so that the increase of the fluorescent signal and the increase of a PCR product are completely synchronous;the PCR reaction system also comprises a PCR buffer solution, a fluorescence labeling Cy3, a dUTP solution, a dGTP solution, a dCTP solution, a PCR protective agent, calcium ions and sterile double distilled water;the PCR reaction system also comprises a forward primer of 10 mu mol/L, and the nucleotide sequence is as follows: GGCACCCAGCACAATGAAGA, respectively;the PCR reaction system also comprises a reverse primer of 10 mu mol/L, and the nucleotide sequence is as follows: ACTCCTGCTTGCTGATCCAC, respectively;the Numb template amount in the PCR reaction system is as follows: based on a 20 mu L reaction system, 6-12 ng of plasmid and 3-8 mmol/L of calcium ion concentration;the quantitative PCR reaction conditions were: 3min at 94 ℃, 20s at 94 ℃ and 30s at 50 ℃ for 40 cycles.Step 4, measuring blood metabolic molecules:measuring the number of metabolic molecules with the molecular weight of 100-3000 daltons in the blood of a patient, and calculating the ratio of the number of the molecules with the signal-to-noise ratio higher than the optimal threshold value to the number of the detected metabolic molecules: mixing blood with nano silicon, sampling the mixed blood on a target plate of a mass spectrum, obtaining data (the precision is 0.001-0.1 dalton) by using a mass spectrometer, wherein the obtained data is the relative number value M of metabolic molecules with the length of more than 100 dalton, and then selecting the number value N of all metabolic molecules with the length of 100-3000 dalton and the signal-to-noise ratio of more than 2.10;step 5, comprehensive judgment:judging the degree of tumor deterioration according to the degree that the content of Musashi2 in the tumor tissue is higher than a normal value and the degree that the content of Numb in the tumor tissue is lower than the normal value, and in combination with the degree that the number of all detected metabolic molecules in plasma is 100-3000 daltons and the signal-to-noise ratio is higher than the normal value;(II) detecting a prognosis stage:step 1, measuring blood Musashi 2:pretreating blood of a postoperative patient, placing the blood into a PCR reaction system, adding a fluorescent dye Sypro and a hot start hTaq enzyme into the PCR reaction system, wherein the fluorescent dye Sypro can be specifically combined with Musashi2 to emit a fluorescent signal, so that the increase of the fluorescent signal and the increase of a PCR product are completely synchronous;the PCR reaction system also comprises a PCR buffer solution, a fluorescence labeling Cy5, a dATP solution, a dGTP solution, a dCTP solution, a PCR protective agent, calcium ions and sterile double distilled water;the PCR reaction system also comprises a forward primer of 9 mu mol/L, and the nucleotide sequence is as follows: AGGTTGAGCCATGCAGTCAT, respectively;the PCR reaction system also comprises a 9 mu mol/L reverse primer, and the nucleotide sequence is as follows: AGGTTGAGCCATGCAGTAGC, respectively;the Musashi2 template amount in the PCR reaction system is as follows: 5-10 ng of plasmid based on a 20 mu L reaction system, and 6-8 mmol/L of calcium ion concentration;the quantitative PCR reaction conditions were: 4min at 90 ℃, 15s at 95 ℃, 35s at 50 ℃ and 40 cycles.Step 2, measuring blood Numb:pretreating blood of a postoperative patient, placing the blood into a PCR reaction system, adding a fluorescent dye Ruby and a hot start hTaq enzyme into the PCR reaction system, wherein the fluorescent dye Ruby can be specifically combined with Numb to emit a fluorescent signal, so that the increase of the fluorescent signal and the increase of a PCR product are completely synchronous;the PCR reaction system also comprises a PCR buffer solution, a fluorescence labeling Cy3, a dUTP solution, a dGTP solution, a dCTP solution, a PCR protective agent, calcium ions and sterile double distilled water;the PCR reaction system also comprises a forward primer of 12 mu mol/L, and the nucleotide sequence is as follows: GGCACCCAGCACAATGAAGA, respectively;the PCR reaction system also comprises a reverse primer of 12 mu mol/L, and the nucleotide sequence is as follows: ACTCCTGCTTGCTGATCCAC, respectively;the Numb template amount in the PCR reaction system is as follows: based on a 20 mu L reaction system, 6-12 ng of plasmid and 5-10 mmol/L of calcium ion concentration;the quantitative PCR reaction conditions were: 3min at 94 ℃, 20s at 94 ℃ and 30s at 50 ℃ for 40 cycles.Step 3, measuring blood metabolic molecules:measuring the number of metabolic molecules with the molecular weight of 100-3000 daltons in the blood of a patient, and calculating the ratio of the number of the molecules with the signal-to-noise ratio higher than the optimal threshold value to the number of the detected metabolic molecules: mixing blood with nano silicon, sampling the mixed blood on a target plate of a mass spectrum, obtaining data (the precision is 0.001-0.1 dalton) by using a mass spectrometer, wherein the obtained data is the relative number value M of metabolic molecules with the length of more than 100 dalton, and then selecting the number value N of all metabolic molecules with the length of 100-3000 dalton and the signal-to-noise ratio of more than 2.10;step 4, prognosis judgment:performing multiple blood detections in a prognosis stage, and comparing the detection result of each time with the detection result of the last time;determining that the content of Musashi2 in the blood of the postoperative patient is higher than the content of Musashi2 in the blood of the last measurement, the content of Numb in the blood of the postoperative patient is lower than the content of Numb in the blood of the last measurement, and determining that all detected molecular weights in the blood of the postoperative patient are 100-3000 daltons, and the ratio of the number of metabolic molecules with the signal-to-noise ratio of more than 2.10 is higher than the last measurement, thus indicating that the disease condition of the patient develops or further worsens; otherwise, the patient is indicated to have improved condition.
- 2. The method of claim 1 for the determination of pancreatic cancer progression by MSI 2-Numb-binding metabolic molecules, wherein in step 2, the working concentration of the fluorescent dye Sypro is 0.3 x to 1 x, and the working concentration of the hot-start hTaq enzyme is 0.03 to 0.1U/μ L; the working concentration of the dATP solution is 50-80 mu mol/L; the working concentration of the dGTP solution is 50-90 mu mol/L; the working concentration of the dCTP solution is 60-80 mu mol/L; the working concentration of the fluorescent label Cy5 is 0.2X to 0.8X.
- 3. The method for determining pancreatic cancer progression by MSI 2-Numb-binding metabolic molecule of claim 1, wherein in step 3, the working concentration of the fluorescent dye Ruby is 0.5 x to 1 x, and the working concentration of the hot-start hTaq enzyme is 0.04 to 0.1U/. mu.l; the working concentration of the dUTP solution is 60-80 mu mol/L; the working concentration of the dGTP solution is 40-70 mu mol/L; the working concentration of the dCTP solution is 50-70 mu mol/L; the working concentration of the fluorescent label Cy3 is 0.4X to 0.7X.
- 4. The method for determining pancreatic cancer progression based on MSI2-Numb in conjunction with metabolic molecules as claimed in claim 1, wherein (two) prognostic phase assay, step 1, the working concentration of the fluorescent dye Sypro is 0.35 x to 1 x, and the working concentration of the hot-start hTaq enzyme is 0.04 to 0.1U/. mu.L; the working concentration of the dATP solution is 90-95 mu mol/L; the working concentration of the dGTP solution is 100-120 mu mol/L; the working concentration of the dCTP solution is 100-120 mu mol/L; the working concentration of the fluorescent label Cy5 is 0.6X to 0.8X.
- 5. The method for determining pancreatic cancer progression based on MSI2-Numb in conjunction with metabolic molecules as claimed in claim 1, wherein (two) prognostic stage detection, step 2, the working concentration of the fluorescent dye Ruby is 0.8 x-1 x, and the working concentration of the hot-start hTaq enzyme is 0.06-0.1U/. mu.L; the working concentration of the dUTP solution is 100-120 mu mol/L; the working concentration of the dGTP solution is 80-100 mu mol/L; the working concentration of the dCTP solution is 80-100 mu mol/L; the working concentration of the fluorescent label Cy3 is 0.5X to 0.8X.
- 6. The method for determining pancreatic cancer progression based on MSI2-Numb in combination with metabolic molecules as claimed in claim 1, wherein the PCR protectant is gelatin, Tween20 or dithiothreitol.
- 7. The method for determining pancreatic cancer progression by MSI 2-Numb-binding metabolic molecules of claim 1, wherein the blood and nanosilica are mixed in a mass ratio of 1: 0.6.
- 8. The method of claim 1 for determining pancreatic cancer progression from MSI 2-Numb-binding metabolic molecules, wherein the ratio of the number of molecules with signal-to-noise ratio above an optimal threshold to the number of selected metabolic molecules detected is calculated using the following set of equations: and obtaining a score ratio which is the number of molecules with the signal-to-noise ratio higher than the optimal threshold value/the number of detected metabolic molecules, namely obtaining the score ratio which is N/M.
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| Title |
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| WEIWEI SHENG 等: "Cooperation of Musashi-2, Numb, MDM2, and P53 in drug resistance and malignant biology of pancreatic cancer", THE FASEB JOURNAL, vol. 31, pages 2429 - 2438 * |
| WEIWEI SHENG 等: "Musashi2 promotes the development and progression of pancreatic cancer by down-regulating Numb protein", ONCOTARGET, vol. 8, no. 9, pages 14359 - 14373 * |
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