Screening method and sensitivity evaluation method of biochip detection system
By establishing amplification threshold and hybridization signal threshold in the biochip detection system, and using PCR real-time fluorescence quantitative analyzer screening and evaluation detection system, the problem of low screening efficiency in the prior art is solved, and fast and efficient screening and sensitivity evaluation are achieved.
Patent Information
- Application Number
- CN202510444716.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-10
- Publication Date
- 2025-07-08
AI Technical Summary
In the prior art, the screening and sensitivity evaluation methods of biochip detection systems are inefficient, which seriously affects the screening efficiency and cycle, and mainly relies on the chip hybridization results as the screening criteria.
The chip hybridization signal amplification threshold is established, and the amplification threshold and hybridization signal threshold are detected by PCR real-time fluorescence quantitative analyzer, detection systems that meet specific conditions are screened, and sensitivity evaluation is performed through gradient dilution nucleic acid templates.
The screening and sensitivity evaluation time of biochip detection system has been greatly shortened, from 30 hours to less than 3 hours, reducing costs and cycles and improving screening efficiency.
Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of biochip detection, and particularly relates to a screening method and a sensitivity evaluation method for a biochip detection system. Background Art
[0002] The main components of a biochip detection system are two parts, reagents and chips. Among them, the production and manufacturing cycle of chips is the longest, generally taking 24 hours to 1 week, while the production of reagents only takes less than 0.5 hours to complete. The experimental process is mainly divided into template amplification, product purification and denaturation, chip hybridization, and scanning analysis, which takes at least more than 6 hours. Therefore, it takes at least 30 hours from the system design and production to the final verification. At present, in the process of biochip development, the screening and sensitivity evaluation methods for detection systems (amplification systems, amplification primers, and chip probes) are relatively limited. Ultimately, the hybridization results of the chips are used as the screening criteria, and the entire process needs to be completed. This method seriously affects the screening efficiency during the preliminary system screening and sensitivity evaluation process, and there is an urgent need for a fast method for screening and sensitivity evaluation of biochip detection systems. Summary of the Invention
[0003] In order to solve the problems in the prior art, the present invention provides a screening method and a sensitivity evaluation method for a biochip detection system, aiming to reduce the overall development or optimization difficulty and cycle of biochip detection-related products.
[0004] The present invention solves its technical problems by adopting the following technical solutions: The present invention aims to provide a screening method for a biochip detection system, comprising the following steps: S1. Establish a chip hybridization signal amplification threshold (hereinafter referred to as amplification threshold): Prepare amplification system A and amplification system B. Amplification system A and amplification system B are respectively added to nucleic acid templates diluted in a 2-fold gradient for amplification and hybridization. After amplification and hybridization of amplification system A with each gradient-diluted nucleic acid template, fluorescence collection is performed, and the CT values and ▲Rn values corresponding to the concentrations of each gradient-diluted nucleic acid template are counted. Since in the same project (the equipment and testing methods used for different detection sites are the same), only one amplification threshold needs to be established; After amplification and hybridization of amplification system B with each gradient-diluted nucleic acid template, it is mixed with the hybridization solution, and then hybridized on a biochip and scanned to obtain a hybridization signal threshold R, and hybridization signal values R1, R2, R3...Rn at the concentrations of each gradient-diluted nucleic acid template are obtained; When Rn < R and Rn-1 ≥ R, the nucleic acid template concentration corresponding to Rn-1 in the CT value and ▲Rn value obtained by amplification of amplification system A with the 2-fold gradient-diluted nucleic acid template is the amplification threshold CT 标准 , ▲Rn标准 ; S2. Screening of the biochip detection system: For different detection systems, use a real-time fluorescence quantitative PCR analyzer to obtain the corresponding CT value and ▲Rn value, and screen the detection system that simultaneously satisfies CT ≤ CT 标准 , ▲Rn ≥ ▲Rn 标准 .
[0005] Among them, the nucleic acid template can be nucleic acid samples such as DNA template, RNA template, plasmid, etc. The biochip can be a microarray chip or a microfluidic chip used for nucleic acid detection. The hybridization signal threshold R is the lowest fluorescence signal value on the biochip that can be interpreted or determined as positive after biochip hybridization. The ▲Rn value is the fluorescence signal value collected after deducting the background signal under PCR conditions, which can accurately reflect the amount of amplification products and is a value read and calculated by a real-time fluorescence quantitative analyzer. The hybridization signal value Rn is the signal value of the negative result calculated by the supporting software after the amplification products are hybridized to the biochip and scanned by a scanner, and is evaluated from multiple groups of data. Usually, different scanning devices and different fluorescence modifications in the system will result in different hybridization signal values Rn. The difference between them is that the ▲Rn value is obtained by a real-time fluorescence quantitative analyzer and analyzes the fluorescence signal of the amplification products in the tube. While the hybridization signal threshold R and the hybridization signal value Rn are obtained by a scanner and analyze the fluorescence signal on the biochip after the amplification products are hybridized to the biochip.
[0006] Amplify and hybridize by 2-fold gradient dilution of the nucleic acid template to obtain the hybridization signal values R1, R2, R3, R4...Rn at different concentrations. When Rn < R and Rn-1 ≥ R, the CT value and ▲Rn value obtained by amplifying the nucleic acid template concentration corresponding to Rn-1 on a real-time fluorescence quantitative PCR analyzer are the amplification thresholds, that is, CT 标准 , ▲Rn 标准 . Then use a real-time fluorescence quantitative PCR analyzer to obtain the corresponding CT value and ▲Rn value through different detection systems. As long as the CT of the experimental group ≤ CT 标准 , ▲Rn ≥ ▲Rn 标准 , then the detection system is suitable for the biochip for which this threshold is established. When multiple groups of detection systems are applicable, calculate their amplification coefficient t = ▲Rn / CT, and select the system with the largest t as the detection system for this site.
[0007] In the present invention, both systems A and B use the same amplification system to amplify the same amplification template and corresponding concentrations (where the excess amplification probes in system A will not affect the concentrations of various substances in the system and the amplification effect of the enzyme), that is, a repetition of conventional PCR. The repeatability coefficient of variation of normal PCR can meet less than 5%, and it is defaulted that their amplification effects are the same.
[0008] Furthermore, both amplification system A and amplification system B include primer F and primer R. The nucleotide sequence of primer F is as shown in SEQ ID NO.1 in the sequence listing, and the nucleotide sequence of primer R is as shown in SEQ ID NO.2 in the sequence listing.
[0009] Furthermore, amplification system A further includes an amplification probe, and the nucleotide sequence of the amplification probe is as shown in SEQ ID NO.4 in the sequence listing.
[0010] Furthermore, the amplification procedures for amplification hybridization of amplification system A and amplification system B respectively in 2-fold gradient-diluted nucleic acid templates are as follows: 55°C, 300 s, 1 cycle; 94°C, 180 s, 1 cycle; 94°C, 15 s, 55°C, 30 s, 72°C, 30 s, 35 cycles.
[0011] Furthermore, in S2, when multiple detection systems all satisfy CT ≤ CT 标准 , ▲Rn ≥ ▲Rn 标准 , then calculate the amplification coefficient t = ▲Rn / CT of each detection system, and select the detection system with the largest t value.
[0012] A method for evaluating the sensitivity of a biochip detection system. Gradient-dilute a nucleic acid template, and after dilution, add it to the selected biochip detection system respectively, and use a PCR real-time fluorescence quantitative analyzer for detection to obtain the CT values and ▲Rn values corresponding to different nucleic acid template concentrations. If CT ≤ CT 标准 , ▲Rn ≥ ▲Rn 标准 are satisfied simultaneously, then the nucleic acid template concentration can be detected on the biochip. When the concentration Cn can be detected and the concentration C(n + 1) cannot be detected, then determine that the concentration Cn is the sensitivity concentration of the biochip detection.
[0013] Furthermore, if the difference between the concentration Cn and the concentration C(n + 1) is too large, take the concentration Cn as the initial concentration, and gradient-dilute it to the concentration C(n + 1) again, and repeat the above detection and determination method to obtain a more accurate sensitivity concentration.
[0014] For the sensitivity evaluation of the biochip of the present invention, a known nucleic acid template concentration C0 is gradient-diluted into C1, C2, C3, C4, C5 ··· Cn, and the gradient dilution multiple can be 0 to 1000 times. By combining the amplification threshold with the use of a PCR real-time fluorescence quantitative analyzer, as long as the CT values and ▲Rn values corresponding to different nucleic acid template concentrations satisfy CT ≤ CT 标准 , ▲Rn ≥ ▲Rn 标准, the concentration of the nucleic acid template can be detected on the biochip. Conversely, if any of the conditions is not met, the concentration cannot be detected. When the concentration Cn is detected and the concentration C(n + 1) is not detected, the concentration Cn can be determined as the sensitivity concentration detected by the biochip. If the difference between the concentration Cn and the concentration C(n + 1) is too large, the concentration Cn can be used as the initial concentration and diluted stepwise to the concentration C(n + 1) again. The stepwise dilution factor can be 0 - 1000 times. Repeat the above detection and determination methods to obtain a more accurate sensitivity concentration.
[0015] The present invention also provides a pair of primer sequences, a chip probe sequence, an amplification probe sequence, and a corresponding plasmid sequence for amplification (the amplification probe sequence is the same as the chip probe sequence, except for different modifications) for establishing the amplification threshold. ① The length of the plasmid sequence can be 120 - 10000 bp. The plasmid length is divided into three regions: the primer region, the amplification region (including the probe region), and the non-amplification region. The region that affects the plasmid length is the amplification region, and its length is determined according to the amplification length of the developed project. The sequence information is shown in Table 1 below.
[0016] Table 1: Sequence Information .
[0017] Compared with the prior art, the beneficial technical effects of the present invention are as follows: 1. The present invention greatly reduces the time for screening the biochip detection system and evaluating the sensitivity. After establishing the amplification threshold, the analysis and judgment can be carried out in less than 3 hours from 30 hours of a single experiment. Only the biochip is needed when establishing the amplification threshold, avoiding the expensive cost brought by the biochip in subsequent verification experiments. The price of the chip ranges from 200 to 20000 yuan per piece.
[0018] 2. The present invention can analyze the detailed performance of the biochip in real-time fluorescence quantitative PCR analysis by establishing the amplification threshold.
[0019] The above description is only an overview of the technical solution of the present invention. In order to understand the technical means of the present invention more clearly, it can be implemented according to the content of the specification. And in order to make the above content, its purpose, features, and advantages of the present invention more obvious and understandable, the following specific embodiments of the present invention are given. Specific Embodiments
[0020] In addition, unless otherwise specifically stated, all kinds of raw materials, reagents, instruments, and equipment used in the present invention can be obtained through market purchase or prepared by existing methods. Example 1
[0021] 1. Establish the amplification threshold: ①The synthesis information of primers, probes, and plasmids is shown in Table 2 below.
[0022] Table 2: Synthesis Information of Primers, Probes, and Plasmids 。
[0023] ②Prepare two systems, amplification system A and amplification system B. Among them, amplification system A is directly analyzed and detected using a PCR real-time fluorescence quantitative analyzer, and amplification system B is used for chip hybridization after amplification with a PCR real-time fluorescence quantitative analyzer. Amplification system A has one more amplification probe than amplification system B. For details, see Table 3 below.
[0024] Table 3: Preparation Table of Amplification System A and Amplification System B 。
[0025] ③For the production of biochips, aldehyde slides are used. The chip probes are diluted to 1 μM and spotted on the aldehyde slides using a spotter to complete the production of biochips for establishing the amplification threshold.
[0026] ④Plasmid dilution and amplification: Dilute the synthesized plasmid by a 2-fold gradient, with dilutions of 2, 4, 8, 16, 32, 64, 128, 256, 512, 1024, 2048, 4096, 8192, 16384, 32768, 65536, 131072, 262144, 524288, and 1048576 times, a total of 20 concentration gradients, labeled as C1, C2, C3... C20. Each of amplification system A and amplification system B is aliquoted into 20 tubes, 15 μL per tube, and labeled as A1, A2... A20, B1, B2.... B20 respectively. Among them, A1 and B1 are added with the plasmid diluted 2 times, A2 and B2 are added with the plasmid diluted 4 times... A20 and B20 are added with the plasmid diluted 1048576 times. Use a PCR real-time fluorescence quantitative analyzer (ABI7500) to collect fluorescence at the CY5 channel for the reagent position of tube A, and count the CT values and ▲Rn values corresponding to different plasmid concentrations of the reagents in tube A as shown in Table 4 below. The amplification program is shown in Table 5 below.
[0027] Table 4: Statistical Table of CT Values and ▲Rn Values of Amplification in Tube A 。
[0028] Table 5: Amplification Program 。
[0029] ④Product hybridization: Aliquot the hybridization solution (4% dextran sulfate, 1% SDS, 3×SSC) into 20 tubes, 13 μL per tube, and label as B1, B2... B20. Take 2 μL of the product from each tube B and add it to the corresponding numbered hybridization solution, mix well and add it to the biochip, and hybridize at 50 °C for 2 h.
[0030] ⑤ Chip cleaning and scanning: First, wash the chip with 0.1% SDS on a shaker for 5 min. After washing, take out the chip and wash it with 0.06×SSC on a shaker for 5 min. Take out the chip and centrifuge it to dry the water. Use a biochip scanner to scan the chip, and the hybridization signal threshold is 400. Read and count the hybridization signal values of different plasmid concentrations as shown in Table 6 below.
[0031] Table 6: Statistical table of hybridization signal values of Tube B 。
[0032] Note: ≤0 means that the site signal ≤ the background signal of the chip and can no longer be detected.
[0033] ⑥ Amplification threshold: Through analysis, it is obtained that for the hybridization signal value R16 = 320 < R = 400 corresponding to B16, and the hybridization signal value R15 = 511 ≥ R = 400 corresponding to B15. Therefore, the amplification threshold is the detection result of plasmid concentration C15 in amplification system A, that is, the CT value and ▲Rn value corresponding to tube A15, ▲Rn 标准 = 450,000, CT 标准 = 29.1.
[0034] Detection system screening: Four groups of primer-probes for detecting corresponding sites were designed, as shown in Table 7 below. It is required that the detection sensitivity can detect 1000 copies / mL. Four groups of amplification systems (No. 1, 2, 3, 4) were prepared with the four groups of primer-probes. The preparation is shown in Table 8. Each group was dispensed into 3 tubes (each system was measured 3 times in duplicate), 15 μL / tube. A standard product diluted to 1000 copies / mL of the detection site was added, 5 μL / tube. Use a real-time fluorescence quantitative PCR analyzer (ABI7500) for detection. The amplification program is shown in Table 5. Select the CY5 channel for fluorescence collection. Calculate the mean values of CT values and ▲Rn values under each group of systems. The results are shown in Table 9 below. In the results, the mean values of CT values and ▲Rn values of No. 1 and 2 can meet CT ≤ CT 标准 ,▲Rn ≥ ▲Rn 标准 。 Calculate their amplification coefficients t. Among them, the system of No. 1 has t1 = 22344, and the system of No. 2 has t2 = 20640, t1 > t2. Therefore, select the detection system of No. 1 as the detection system for this detection site.
[0035] Table 7: Information table of primer-probes 。
[0036] Table 8: Preparation table of four groups of systems 。
[0037] Table 9: Statistical table of results 。
[0038] Example 2: Actual sensitivity evaluation of the detection system screened in Example 1 (detection system No. 1) Prepare the system according to the formula in Table 8 and dispense into 9 tubes, 15μL / tube. Dilute the standard concentration of 1000 copies / mL by 2-fold gradient to 500 copies / mL, 250 copies / mL, and 125 copies / mL, respectively, and add them to the above-dispensed system, 5μL / tube, 3 tubes for each concentration, and use PCR real-time fluorescence quantitative analyzer (ABI7500) for detection. The amplification program is shown in Table 5. Select CY5 channel for fluorescence collection, calculate the mean of CT value and ▲Rn value of each group of data, and the results are shown in Table 10 below. Among them, CT of 250 copies / mL = 30.2>CT 标准 =29.1, ▲Rn=410,000<▲Rn 标准 = 450,000, and CT at 500 copies / mL = 28.2 < CT 标准 =29.1, ▲Rn=550,000>▲Rn 标准 = 450,000, so the actual achievable sensitivity concentration of this detection site under the screened detection system is 500 copies / mL.
[0039] Table 10: Results statistics .
[0040] Example 3: The detection system (detection system No. 1) selected by Example 1 and Example 2 and the evaluated sensitivity concentration (500 copies / mL) were retested using the corresponding biochip The primer sequence for synthetic biochip is consistent with the primer probe sequence No. 1 selected in Table 7, wherein the 5' end of the R primer (downstream primer) needs to be fluorescently modified, the 5' end of the chip probe sequence is modified with NH2 instead, and the 3' is not modified, see Table 11 below.
[0041] Table 11: Biochip primer probe information .
[0042] For biochip production, aldehyde-based glass slides are used, and the chip probes are diluted to 1 μM and spotted on the aldehyde-based glass slides using a spotter to complete the production of the biochip.
[0043] Amplification hybridization: Prepare the amplification system for chip hybridization. The formulation information is shown in Table 8. Aliquot it into 3 tubes, 15 μL per tube. Dilute the standard product of the detected locus to 500 copies / mL and add it to the above amplification system, 5 μL per tube. Use a real-time fluorescence quantitative PCR analyzer (ABI7500) for amplification. Aliquot the hybridization solution (4% dextran sulfate, 1% SDS, 3× SSC) into 3 tubes, 13 μL per tube. Take 2 μL of each amplified product and add them to the hybridization solution with corresponding numbers respectively. Mix well and add them to the prepared chip. Hybridize at 50 °C for 2 h. After hybridization, first wash with 0.1% SDS on a shaker for 5 min. After washing, take out the chip and wash it with 0.06× SSC on a shaker for 5 min. Take out the chip, centrifuge to dry the water, and use a biochip scanner to scan the chip. The hybridization signal values of 3 replicates are statistically shown in Table 12 below. The signal values of the three times are all greater than the hybridization signal threshold R = 400. Retest proves that the detection system screened by this method and the evaluated sensitivity concentration can be detected on the biochip.
[0044] Table 12: Result statistics 。 Example 4
[0045] During the screening process of a 10-locus project system, it was statistically found that the system was adjusted 6 times in total. The statistical results of the screening cost cycle of the system using this method are shown in Table 13; the statistical results of the screening cost cycle of the system using the conventional method after conversion are shown in Table 14, in which 6 chip designs and productions are required. By comparing the two system screening methods, it can be seen that the screening cycle of this method for the 10-locus system is 26.7% of the conventional method, and the cost is 28.2% of the conventional method. Since in the same project (the equipment and inspection methods used for different detection loci are the same), only one amplification threshold needs to be established, so as the number of times of locus or system adjustment increases, the benefits brought by this screening method are better.
[0046] Table 13: Statistical results of the screening cycle and cost of this method system 。
[0047] Table 14: Statistical results of the screening cycle and cost of the conventional method system 。
[0048] The serial numbers of the above embodiments of the present invention are only for description and do not represent the advantages or disadvantages of the embodiments.
[0049] The embodiments of the present invention have been described above. However, the present invention is not limited to the above specific embodiments. The above specific embodiments are merely illustrative rather than restrictive. Under the inspiration of the present invention, those of ordinary skill in the art can also make many forms without departing from the spirit of the present invention and the scope protected by the claims. All of these fall within the protection scope of the present invention.
Claims
1. A screening method for a biological chip detection system, characterized in that, It includes the following steps: S1. Establish the amplification threshold of chip hybridization signals: Prepare amplification systems A and B. The amplification systems A and B are respectively added to nucleic acid templates diluted in a 2-fold gradient for amplification hybridization. After the amplification hybridization of the amplification system A with each gradient-diluted nucleic acid template, fluorescence collection is carried out, and the CT values and ▲Rn values corresponding to the concentrations of each gradient-diluted nucleic acid template are counted; After the amplification hybridization of the amplification system B with each gradient-diluted nucleic acid template, it is mixed with the hybridization solution, and then hybridized on the biochip and scanned to obtain the hybridization signal threshold R, and the hybridization signal values R1, R2, R3...Rn at the concentrations of each gradient-diluted nucleic acid template are obtained; When Rn < R and Rn-1 ≥ R, the nucleic acid template concentration corresponding to Rn-1, the CT value and ▲Rn value obtained from the amplification system A and the nucleic acid template amplified by 2-fold serial dilution are the amplification threshold CT 标准 , ▲Rn 标准 ; S2. Screening of the biochip detection system: For different detection systems, use a real-time fluorescence quantitative PCR analyzer to obtain the corresponding CT value and ▲Rn value, and screen the detection system that simultaneously satisfies CT ≤ CT 标准 , ▲Rn ≥ ▲Rn 标准 .
2. The screening method of a biochip detection system according to claim 1, characterized in that: Both the amplification systems A and B include primer F and primer R. The nucleotide sequence of primer F is as shown in Sequence Listing SEQ IQ NO.1, and the nucleotide sequence of primer R is as shown in Sequence Listing SEQ IQ NO.
2.
3. The screening method of a biochip detection system according to claim 2, characterized in that: The amplification system A further includes an amplification probe, and the nucleotide sequence of the amplification probe is as shown in Sequence Listing SEQ IQ NO.
4.
4. The screening method of a biochip detection system according to claim 3, wherein: The amplification procedures for the amplification hybridization of the amplification systems A and B with the nucleic acid templates diluted in a 2-fold gradient are respectively: 55°C, 300 S, cycle 1 time; 94°C, 180 S, cycle 1 time; 94°C, 15 S, 55°C, 30 S, 72°C, 30 S, cycle 35 times.
5. The screening method of a biochip detection system according to claim 1, characterized in that: In S2, when multiple detection systems all satisfy CT ≤ CT 标准 , ▲Rn ≥ ▲Rn 标准 , then calculate the amplification coefficient t = ▲Rn / CT of each detection system, and select the detection system with the largest t value.
6. The sensitivity evaluation method of the screening method of a biochip detection system according to any one of claims 1-5, characterized in that: Gradiently dilute the nucleic acid template, and after dilution, add it to the screened biochip detection system respectively. Use a real-time fluorescence quantitative PCR analyzer for detection to obtain the CT values and ▲Rn values corresponding to different nucleic acid template concentrations. If both CT ≤ CT 标准 , ▲Rn ≥ ▲Rn 标准 are satisfied, then the concentration of this nucleic acid template can be detected on the biochip. When the concentration Cn can be detected and the concentration C(n + 1) cannot be detected, then the concentration Cn is determined as the sensitivity concentration for the detection of this biochip.
7. The sensitivity evaluation method according to claim 6, wherein: If the difference between the concentration Cn and the concentration C(n + 1) is too large, use the concentration Cn as the initial concentration, and dilute it again to the concentration C(n + 1) in a gradient, and repeat the above detection and determination method to obtain a more accurate sensitivity concentration.