TWM419106U - Gene group test structure - Google Patents

Description

M419106 iiMf 100 V. New description: [New technical field] This creation is about a gene group detection structure, especially one that improves the disadvantages of the original nylon diaphragm operating platform. In particular, it refers to the establishment of a new low-cost WEnCA ( The Weighted Enzymatic Chip Array) can be combined with automated operation to reduce inspection time and reduce human error, so as to overcome the bottleneck in the commercialization process of dog and wafer detection technology. [Prior Art] Analysis of gene transition performance (〇verexpressi〇n) has led to basic advances and clinical advances in disease diagnosis. The technology for studying gene transition performance includes Northern Blot, Reverse Transcription Polymerase Chain Reaction (RT-PCR), and Real-Time Polymerase Chain Reaction (Real_Time pcr). Among them, Northern Blot is too sensitive for the operation steps, and the amount of samples required is too large, so it is limited to the research operation 'can not be practically applied to clinical diagnosis. As for RT pCR and Real_Time PCR, because of the simple operation steps, it is widely used in the application of single gene detection, such as detection of hepatitis virus and identification of infectious pathogens. However, although the creation of PCR sequences is the greatest experiment in the whole, most PCR technologies still have specific problems in common. The main problems include: a series of pollution, false positives for transitional sensitive reconnaissance, such as foggy DNA or Previous sample residues; the second line RT_PCR is only considered to be semi-quantitative because it is difficult to control the efficiency of sequence amplification when comparing different samples; and the three lines are interfered by the inter-primer interaction (Annearling), regardless of RT_pCR or 3 M419106 ^yjiuyi^y eal TimePCR is widely used only for the detection of single gene markers, and the shortcomings of 'WPCR-related technology operations are time-consuming, costly and costly.

With the rapid development of biotechnology, BioCheng# has gradually been valued in clinical medical diagnosis or evaluation of drug efficacy. Previous studies of this work have developed and evaluated a MembraneArray method that can be used. For cancer diagnosis, the expression level of a plurality of mRNA-labeled primers is simultaneously detected in a blood sample (Peripheral Blood). The expression level of the molecular markers was evaluated by RT-PCR and nylon membrane wafers, and the data was obtained from RT_pcR and nylon membrane wafers, and forked in linear regression analysis, showing a high correlation between the results of the two methods (γ=0.979 , Ρ <0.0001) β In addition, Weighted Chemoluminescent Gene Wafers with Related Derivative Techniques (Weighted Chemiluminescent)

Membrane Array, WCHMA) The role of Target Therapeutic Drug in patients with lung cancer (Lung Cancer) The abnormal condition of the subject K-ras has also been accepted for publication in the Journal of Lung Cancer.

)〇. y. 2 9#jEl ^年月曰&> 2011/9/29 Supplement Although the application of nylon membrane wafers in molecular diagnostics and pharmacodynamic evaluation has been reported many times, however, due to the original nylon membrane In the interpretation method used for the wafer, under the concept that each gene is equivalent in the importance of a specific disease, the detection specificity is not easily improved after reaching a certain level. In addition, due to the high cost of the Digoxigenin system used in the Colorimetric Biochip operating platform, the cost of testing is too high, coupled with the high technical threshold of wafer operation, even if it has been developed and Assessing mature diagnostic wafers is still not readily available for clinical medical applications. Therefore, the general practitioners cannot meet the needs of the user in actual use. 4 M4191〇6 [New Content] The main purpose of this creation is to overcome the above problems encountered in the prior art and to provide a WEnCA-Chipball operating platform that is fast, accurate, sensitive, low-cost, extensively analyzed and easy to integrate with automation. The secondary objective of this creation is to provide a structure that is easy to automate in various experiments without the need for a large centrifuge.

Another object of the present invention is to provide a structure in which magnetic beads containing a p〇ly T primer are used to extract a higher purity of the mRNA, and the background value of the wafer after the reaction is lowered with high accuracy. A further object of the present invention is to provide a biotin-avidin (Biotin-Avidin) instead of Digoxigenin, which is not only low in cost but also uses diaminobenzidine (DAB) as a color former. The stability is also high, so that the color is easy to preserve the structure of the structure.

A further object of the present invention is to provide a method for determining the positive value of the genetically weighted calculations, in conjunction with the ROC Curve, and the accuracy of the clinical test results to more accurately assist the structure of the disease diagnosis. For the purpose of the above, the present invention is a gene group detection structure, which comprises at least a casing for containing the first, second and third bearing portions; a genetic detecting wafer disposed in the first bearing portion; a sample pretreatment unit in the second carrier; a hybrid reaction zone disposed in the third carrier; a wafer coloring unit disposed in the housing; and an analysis disposed on one side of the housing Interpretation unit. In an embodiment of the present invention, the casing may be provided with an induction motor coupled to the first, second and third load-bearing portions. In one embodiment of the present invention, the genetic detection wafer system selects one with cancer 5

The relevant target gene group will contain a plurality of target gene gene groups k - 4 control groups (Internal Control) and a blank control group (Blankc 〇 mr 〇 1) three repeating lattices on a nylon membrane, formed in The genetic detection wafer is coated with a calibration specific sequence, and the genetic detection wafer is a screening wafer having a construct for constructing a complete disease diagnosis, efficacy evaluation or genetic pathogenicity gene. In one embodiment of the present invention, the target gene group is given an individual weighted score of four grades, and when the gene point exhibits a transitional expression in more than 80 cancer tissues, the weighting value is 4; The transition performance of 70 to 80 cancer tissues showed a 'weighted value of 3; when the gene point showed a transitional performance in 60 to 70 cancer tissues, the weighted value was 2; and when the gene point was 50 to 6 癌 cancer tissue The transition performance is presented in the middle, with a weighted value of 1. In one embodiment of the present invention, the target gene group is ATP2A2, ATP6V0B, CXCR4, CYR61, RAP1B, RPL30, BMPR2, CALM2, DVL3, E2F4, SLC25A5, SPP, CEBPB, CLSTN1, ETS1 'H2AFZ' TAF12 'TBX19 'C0L4A1 'CXCL11' L1CAM and LRP In one embodiment of the present invention, the internal control group is β•actin (β-actin) ° In one embodiment of the present invention, the pre-processing unit of the sample The mRNA in the sample was purified by magnetic beads, and the mRNA was reverse-transcribed into cdNA, and then labeled as a probe with an enzyme. In one embodiment of the present invention, the test system can be blood, body fluid, cell culture, and tissue cells. In one embodiment of the present invention, the hybrid reaction zone is used to hybridize the probe to a 5H gene detection wafer. M419106

In one embodiment of the present invention, the wafer coloring unit is configured to perform a color reaction of the hybrid probe on the genetic detection wafer with a diaminobenzidine (DAB) color former. Development). In an embodiment of the present invention, the analysis interpretation unit is internally provided with an analysis software, an image capture mode, and a data transmission mode for capturing the image of the gene detection chip, and coloring The image results after the reaction are automatically analyzed. The detected values obtained after the analysis are weighted by the gene, and the individual weighted scores are given according to the importance of each gene for the occurrence of disease formation or drug resistance, and then multiplied by the positive reaction gene points. The total score of the wafer is obtained by weighting the gene points. In one embodiment of the present invention, the analysis interpretation unit calculates the gene detection by means of a Receiver Operating Characteristic Curve (ROC Curve). The positive value of the wafer is (p〇sitive cut〇ff value) » In one embodiment of the present creation, the positive interpretation standard value is 2〇±2〇%. In one embodiment of the present invention, the detection limit of the gene group detection structure is 20% of 2.4cell/C.C Blood soil. [Embodiment] Please refer to the "Figures 1, 2, 3 and 4", which are the schematic diagram of the three-dimensional appearance of the creation and the decomposition diagram of the creation. Schematic diagram of the arrangement of gene positions. As shown in the figure: This creation is a gene group detection node IMM419106 (test CA-Chipball) 10 Ο, which includes at least the - casing 6 (i, - genetic detection wafer 1 G two specimens secret unit 2 Q, - the hybrid reaction zone 30, the wafer coloration early 704 0 and the analysis interpretation list ^ 5 〇. The above-mentioned housing 6 〇 system activities accommodate the first carrier portion 6 〇丄, the second carrier portion 6 0 2 and the third carrying portion 6 〇3, and the casing 6 可 can be connected to the first, second and third receiving portions Λ b υ 1, 6 〇 2, 6 Ο 3

Motor 6 G 4, in addition to the user can manually open and close the second, third and third load - P6 G 1, 6 Q 2, 6 0 3 ' can also be activated automatically with the induction motor 6 〇 4, The first, second and third load-bearing portions 6 are 0 0 3°. The genetic test wafer 1 is disposed in the first load-bearing portion 6 〇i and is covered with a label, which has a built-up integrity, Screening of wafers for efficacy evaluation or genetic disease-causing genes.

The pre-processing unit 2 is disposed in the second carrying portion 6 〇2, and the sample can be added to the _ _ _ _ _ _ _ _, through the addition of magnetic beads (MagneticParticles) and intracellular Combining the nucleic acid on the magnetic beads with the lysis buffer, and then eluting (Elute) the mRNA on the magnetic beads, and purifying the obtained A, after reverse transcription into cDna, and then labeling the probe with the enzyme (Prcbe), wherein the sample is injected into blood, body fluid, cell culture, and tissue cells. The hybrid reaction zone 30 is disposed in the third bearing part 6 〇3 for reacting the probe with the detection wafer; L 〇 hybrid (Hybridizatk) n), and unreacted The needle is washed from the wafer. The wafer coloring unit 40 is set in the casing 6 (), and the rib is used to detect the gene of the aa sheet 1 and the hybrid probe is added with diaminobenzidine 8

(Diaminobenzidine, DAB)

Development) Coloring reaction of coloring agent (Color) The analysis reading unit 50 is set on the surface of the casing 60. It has built-in analysis software image contact mode and data miscellaneous mode for image mode. The image of the Ui 〇 color reaction was taken, and the image of the color reaction was automatically analyzed by the analysis software, and the detected value obtained by the analysis was calculated by gene weighting. The importance of the occurrence of drug resistance or drug resistance is given to the side weighted score, and the total score of the wafer (lbtals(3)re) is obtained by multiplying the positive reaction gene point by the weighted value of the gene point. The above-mentioned system constitutes a new gene group detection structure丄〇〇 This creation focuses on the shortcomings of the original nylon diaphragm operating platform, and the design improvement strategy's, for example, give different degrees of weighting (LungCaneerRef) to the importance of each target gene on the wafer during interpretation, and biotin-resistant The Biotin-Avidin color system replaces the original Dig〇xigenin system and establishes a new WEnCA (Weighted EnzymaticChipAiTay) operation. Because this structure is easy to combine with the fluid control platform, the automation system can't greatly reduce the detection time and reduce the error caused by the difference of human operation, thus breaking the bottleneck faced by the wafer inspection technology in the commercialization process. The utility of the group detection structure in clinical medical testing, in a preferred embodiment, is to obtain a blood sample of a colorectal cancer (CRC) patient confirmed by 100 clinical pathology departments, which was previously constructed. Activated K_ras Detection Chip (Activated K_ras Detection Chip) 1 〇Use the nylon membrane and the structure i 〇〇 (ie WEnCA-Chipball) to detect the K-ras pathway-related gene in the specimen (K-ras M419106 1y£i^

Pathway Related Genes) transition performance (〇verexpressj〇n) I situation - '?'"',·'''^ and comparison of sensitivity (Sensitivity), specificity (Speciflcity) and accuracy (Accuracy), and analysis When the detection platform of the structure 1 and the original nylon diaphragm is used in clinical application, the operation time and the required cost are different, so as to further establish the development potential of the structure in clinical application. The above-described activated K-ras oncogene detection wafer i 〇, as shown in Fig. 4, selects a target gene group related to colorectal cancer, and includes a target gene group containing 22 target genes and an internal control group (Intemal). C〇ntr〇l) and a blank control group (BlankControl) three repeating lattices on a nylon membrane, where 'the target gene group is ATP2A2, ATP6V0B, CXCR4, CYR61 ' RAP1B ' RPL30 > BMPR2 > CALM2 ' DVL3 ' E2F4 ' SLC25A5, SPP BU CEBPB, CLSTN1, ETS1, H2AFZ, TAF12, TBX19 ' COL4A CXCL11, L1CAM and LRP and the internal control group is β-actin (β-actin). The kisses are shown in Tables 1 and 5, which are the scale tables for the transition of the gene group in the cancer tissue, and the characteristics of the receiver's operation characteristics. As shown in the chart: In the analysis of the weighted luminescent wafer reaction, the creation firstly stipulated the proportion of the 22 gene points on the wafer in the transitional performance of one of the cancer tissues with activated K-ras mutations (cancertissue) and It is divided into four grades. As shown in the following Table 1, the gene points show transitional performance in more than 8 cancer tissues, with a weighted value of 4; and a transitional performance in 70 to 80 cancer tissues, the weight value is 3; transitional performance only in the 6 〇 ~ 7 癌 cancer tissue gene point 'weighted value of 2; if only 5 〇 ~ 6 〇 cancer tissue measured in the transitional gene point, the weight value is 丨. After multiplying the number of genes in the positive reaction on each of the two sets of 丨(8) reactions, multiplying the weighted value, first calculate each 10 M419106 10)·near 9.19 θ θ, the total score of the wafer after the daily replenishment Then, it is also based on whether the tissue actually has a -0 point of mutation - as a standard reference value. The Receiver Operating Characteristic Curve (ROC Curve) 6 ' is shown by biostatistical analysis. As shown in Fig. 5, it can be seen that when the value of the wafer positive reaction (Cutoff Value) is determined to be 20 The detection result of the wafer in the tissue has a sensitivity of up to 96% and a specificity of 97%. Table I

Gene name Transition ratio - (overexpressi onrate(%)) Gene name Transition ratio (overexpressi on rate (%)) Gene name (over the name of the overexpressi on rate (%)) ATP2A2 83 SLC25A5 74 H2AFZ 65 CXCR4 81 CALM2 75 TBX19 64 RAP1B 89 E2F4 78 COL4A1 51 ATP6V0B 90 SPP1 72 L1CAM 53 CYR61 86 CEBPB 66 CXCL11 54 RPL30 92 ETS1 68 LRP1 57 BMPR2 76 TAF12 64 Total Score 58 DVL3 79 CLSTN1 68 Please See "Figure 6" for a schematic of the wafer image after the reaction of the created cancer tissue. As shown in the figure: the wafer image after the reaction of the above cancer tissue 'cancer tissue with K-ras mutation, referred to as CAKM; and with native κ- The cancer tissue with wild type K-ras is called CAKWT. The total scores of CAKM-1 and CAKM-2 after reaction were 36 and 32, respectively>2〇, so the wafer reaction was positive (Positive); the total score after CAKWT-1 and CAKWT-2 reaction was 8 respectively. And 6, both < 20, so the wafer reaction 11 prepared 9·η positive Α gt > the result is negative (Negative). ---~ Please refer to the figure 7 of r, which is a schematic diagram of the interpretation results of the detection limit values of this creation. As shown in the figure: In the interpretation of the experimental results of the detection limit of the artificial gene group detection structure, it can be seen that when 5C C whole blood is added to 100, 50, 25 and 12 activated mutations κ- The total score of cancer cells of the activated mutant K-ras is greater than 2〇. When the number of cells added is 6, the total score is equal to 8 and less than 20, so the detection limit is about 2.4cell/CC Blood. It is more sensitive than the 5cdl/cc Blood measured by the color-coded wafer. Please refer to Figure 8 for the linear regression statistical analysis of this creation. As shown in the figure: This author further analyzes the consistency of the results obtained by the above two methods (ie, this creation and the prior art). After linear regression statistical analysis, it can be seen that the 'r value is 0.86' has statistically significant consistency. It can be seen that the creation has higher sensitivity and better accuracy than the prior art. According to this, this creative department can provide a platform that is fast, accurate, sensitive, low-cost, large-scale analysis and easy to integrate with automation. It can perform rapid biopsy detection and analysis, and achieve the goal that cannot be achieved by traditional biomedical testing. Since this creation does not require a large centrifuge, it is easy to operate in various experiments and is easy to automate, and the magnetic beads containing the P〇ly_T primer are used to extract the higher purity of the mRNA, so that the background value of the wafer after the reaction is lowered, and the accuracy is improved. high. In addition, this work replaces Digoxigenin with Biotin-Avidin, which not only uses the enzyme at low cost, but also has high stability with DAB as a coloring agent, and the color is easy to preserve. Moreover, this creation is also genetically weighted in the final result interpretation. The method of determining the calculated value, together with the ROC Curve, determines the positive interpretation standard. The above clinical test results show that the accuracy of the structure can more accurately assist in the diagnosis of the disease. M419106 · Year Vljf 2011/9/29 Completion. Therefore, this creative department can improve the sensitivity, specificity and accuracy of the diagnosis at the same time. Plus, if combined with the automatic operating system, the system time can be greatly reduced. This creation is a gene group detection structure. Effectively improve the shortcomings of the application, it can provide a fast, accurate, sensitive, low-cost, large-scale analysis and easy to automate WEnCA-ChipbaU operating platform, which can be fast

Low and reduce human error to overcome the bottleneck in the commercialization process of wafer inspection technology. The detection and analysis of biological samples, which is the target that cannot be achieved by traditional biomedical testing, makes the creation of this creation more progressive, more practical, and more in line with the needs of users. It has indeed met the requirements for the creation of patent applications.提出 Submit a patent application in accordance with the law. However, the above-mentioned ones are only the preferred embodiments of this creation. When it is not possible to limit the present cake, the simple equivalent variation of the special fiber and the new manual content should be It is still covered by this creation patent. [Simple description of the diagram] Figure 1 is a schematic diagram of the three-dimensional appearance of the creation. Figure 2 is an exploded view of the creation. Figure 3 is a schematic diagram of the structure of the gene group detection of the present creation. Fig. 4 is a schematic diagram showing the arrangement of gene positions of the present gene detection wafer. Figure 5 is a schematic diagram of the receiver operating characteristic curve of the present creation. Figure 6 is a schematic diagram of the wafer image after the reaction of the created cancer tissue. Figure 7 is a schematic diagram of the interpretation results of the detection limit values of the present creation. Figure 8 is a schematic diagram of linear regression statistical analysis of this creation. 13 M4-19106

[Description of main component symbols] Gene detection structure 1〇〇Gene detection wafer 1〇Pretreatment unit 2 0 Hybrid reaction zone 3◦ Wafer coloring unit 40 Analysis interpretation unit 5 〇Recipient operation characteristic curve 6 Case 6 0 first carrying portion 6 01 second carrying portion 602 third carrying portion 603 induction motor 6 0 4

14

Claims (1)

M419106 Sixth, the scope of application for patents: 1 · A gene group detection structure, comprising: a second bearing portion and a first casing, the activity is accommodated with a first bearing portion of the three carrying portions; a genetic testing chip, is disposed in the first bearing In the second part of the sample, the pre-processing unit is set in the second carrier, and the magnetic beads are used to purify the mRNA in the sample. After the mRNA is reverse-transcribed into cDNA, the enzyme is labeled as a probe ( a heterozygous reaction zone is disposed in the third carrier for synthesizing the probe with the genetic detection wafer; a wafer coloring unit is disposed in the housing The color probe is used to add the diammonium benzidine DAB coloring agent to the probe on the genetic detection wafer, and an analytical interpretation unit is disposed on one side of the casing. The built-in analysis software and image capture mode and data transmission mode are used to capture the image after the color detection reaction of the gene, and automatically analyze the image result after the color reaction, and analyze the image. The detection value is weighted by gene, and each weighted score is given according to the importance of each gene for disease formation or drug resistance, and the total score of the wafer is obtained by multiplying the weight of the gene point by the positive reaction gene point (Total) Score) 〇2. The gene group detecting structure according to claim 1, wherein the casing may be provided with an induction motor 〇15 3 connected to the first, second and third carrying portions. The gene group detection structure described in item i of the patent range ^^ The gene detection chip system is selected - the gene group with the cancer paste will contain the target gene group of various target genes and - internal control group (5) And a blank control group (BlankControl) three repeats 'dot on a nylon membrane to form a labeled specific sequence on the genetic detection wafer, and the genetic detection wafer system has a constructed complete disease diagnosis, efficacy Screening for wafers for evaluation or hereditary pathogenic genes. 4. According to the gene group detection structure described in the third application of the patent garden, the weighted score of the target group 0 is divided into four grades. When the gene point shows transitional performance in more than 80 cancer tissues The weighted value is 4; when the gene point shows a transitional performance in 70~8G enemy weaves, the weighted value is 3; when the gene point shows a transitional performance in 6G~70 cancer tissues, the weighting value is 2; The gene point showed a transitional performance in 50 to 60 cancer tissues with a weighted value of 1. 5 according to the gene group detection structure described in claim 3, wherein the target gene group is ATP2A2, ATP6V0B, cxaw, CYRM, RAP1B, RPL30, BMPR2, CALM2, DVU, E2R _Α5, SPP1, CEBPB, CLSTm, ETS, H2AFZ, TAF12, TBX19, COL4A1, CXCLU, L1CAM, and LRP1. 6. The gene group detection structure according to claim 3, wherein the internal control group is β-actin (J3_actin). 7. The gene group detecting structure according to the first aspect of the patent application, wherein the test system may be blood, body fluid, cell culture, and tissue cells. * According to the gene group detection structure described in the first application scope of the patent application, wherein the analysis interpretation unit reads the receiver operating characteristic scale (heart _ 〇 ating 2011 2011 2011 / / 2011 2011 2011 2011 2011 RO RO RO RO RO RO RO RO RO RO RO RO RO RO The positive cutoff value of the wafer. 9. The gene group detection structure according to item 8 of the patent application scope, wherein the positive interpretation standard value is 20±20%. 1 0 · According to the gene group detection structure described in the scope of claim 1, wherein the detection limit of the detection structure of the gene group is 2.4cell/C.C Blood±20% 〇 M419106 Ot 0«? tr laHdonspcIqEBipolm^fQ· i f c 2SM ) long* (daHildsw) «^g* aax.cssic<^ «^uiBldaasiuv s-^dKH ¢^9^^-^ ,r 11 (ιυ£9-ρ£Μ6一°Μ Μ=Μ > leullelnulsouolssds 9υβω Η-ν ^*名^tfya·6一(OJOUSISOi)** p («""•slpuupdds'-o S°U96S6JB1) Λν (0)=>utM!M9M*-u^" pue u6"9T,2c9E6eJ=e,ec,0o) Provincial umbrella female «*- *耷^®^ ϋ (9UBJqE9ui uu UOBds) ^«-^^^«u ("lueuisejjsua13)''-''*0^ i(c,0s,§,?,^7e,a,dizl5"5 · (νΝαοβ-ΙΕβιυιδιω)§, §, fp (3SOO> eji ) ((1 s>) Iq elof ) e9mv 21 ss) (din? 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Designated representative figure: ( a) The representative representative of the case is: (2) Figure (2) The symbolic symbol of the representative figure is briefly described: Gene detection wafer 10 Pre-processing unit 2 0 Hybrid reaction zone 3 0 Wafer coloring unit 4 0 Analysis interpretation Unit 50 0 Housing 6 0 First bearing part 601 Second carrying part 6 0 2 Third carrying part 6 0 3 Induction motor 6 0 4