PCR detection fluorescence signal crosstalk decoupling method
By generating crosstalk correction coefficients, directly calculating and deducting crosstalk signals between fluorescence channels, the problem of signal crosstalk in PCR instruments is solved, and efficient and low-cost signal decoupling and detection accuracy are achieved.
Patent Information
- Application Number
- CN202510511374.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-23
- Publication Date
- 2025-07-29
AI Technical Summary
The signal crosstalk problem between fluorescence channels in existing PCR instruments leads to a decrease in detection accuracy, and the traditional spectral characteristic analysis methods are complex in operation, high in cost, and correction accuracy are affected by environmental factors.
By generating the crosstalk correction coefficient, the crosstalk correction coefficient is calculated directly and subtracted from the PCR amplification curve of the fluorescent channel that provides crosstalk and accepts crosstalk, and the crosstalk correction coefficient is calculated using a linear fitting method.
Simplifies operation steps, reduces costs, improves the accuracy of signal decoupling and detection accuracy, and is suitable for complex many-to-one crosstalk situations.
Smart Images
Figure CN120388613A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of molecular biological detection, and particularly relates to a method for decoupling fluorescence signal crosstalk in PCR detection. Background Art
[0002] Real-time fluorescence PCR technology has become the core means of nucleic acid detection in the field of molecular biology with its advantages of high sensitivity, specificity, and quantitative detection, and is widely used in many scenarios such as clinical diagnosis, disease monitoring, and scientific research exploration.
[0003] Currently, most common PCR instruments on the market are designed with 4 to 6 fluorescence channels. However, this design cannot meet the increasing high-throughput detection requirements. The core bottleneck lies in the inherent limitations of the response bands of fluorescence sensors and the relatively close excitation / emission wavelengths between different fluorescence channels, resulting in signal crosstalk problems. These problems make it difficult to increase the number of single-tube fluorescence channels of PCR instruments, and false positives (i.e., signals appear in channels where there should be no signals, resulting from ineffective removal of crosstalk signals) or ghosts (i.e., excessive removal of signals, misjudging real signals as interference and weakening them) still occur in the products on sale, affecting the accuracy of detection. Therefore, how to solve the signal crosstalk problem of different fluorescence channels has become the key point for promoting the continuous progress of fluorescence PCR technology.
[0004] Currently, the main methods for solving the fluorescence signal crosstalk problem are mathematical correction methods based on spectral characteristic analysis. This method corrects fluorescence crosstalk by calculating the excitation and emission spectral ratios. First, perform separate spectral measurements on each fluorescent dye used to establish a spectral database for each fluorescent dye; then use a spectrometer to measure the emission spectra of the mixed dyes at each excitation wavelength to obtain the spectral data of the mixed dyes; based on the spectral database and the spectral data of the mixed dyes, calculate the crosstalk ratio of each dye to other dyes through mathematical formulas to construct a crosstalk matrix; finally, correct the measured signals of each dye according to the crosstalk ratio to remove the crosstalk components in the fluorescence signals. This method has cumbersome experimental operations, requires time and effort to establish a spectral database and measure the spectra of mixed dyes, and has high experimental costs; the calculations are complex and are easily interfered by various factors such as environmental temperature and solution properties, affecting the accuracy of correction. Summary of the Invention
[0005] Aiming at the deficiencies of the existing technology, the present invention provides a method for decoupling fluorescence signal crosstalk in PCR detection, which generates a crosstalk correction coefficient according to the PCR amplification curves of the channels providing crosstalk and receiving crosstalk.
[0006] To achieve the above technical objectives, the present invention adopts the following technical solutions:
[0007] A method for decoupling fluorescence signal crosstalk in PCR detection is provided, including the following steps:
[0008] S1: Determine the fluorescence channels where fluorescence crosstalk occurs, including the fluorescence channel providing crosstalk and the fluorescence channel affected by crosstalk;
[0009] S2: Conduct a PCR amplification reaction experiment to generate PCR amplification curves for the fluorescence channel providing crosstalk and the fluorescence channel affected by crosstalk respectively, and calculate the crosstalk correction coefficient between the two fluorescence channels according to the PCR amplification curves;
[0010] S3: Conduct a PCR amplification reaction detection to obtain the fluorescence data of each fluorescence channel;
[0011] S4: According to the crosstalk correction coefficient and the fluorescence data of each fluorescence channel, subtract the corresponding crosstalk of the fluorescence channel providing crosstalk from the fluorescence data of the fluorescence channel affected by crosstalk to obtain the net fluorescence data after decoupling the fluorescence channel crosstalk.
[0012] Preferably, for step S1, to determine the fluorescence channels where fluorescence crosstalk occurs, including the fluorescence channel providing crosstalk and the fluorescence channel affected by crosstalk, the following steps are included:
[0013] S11: For each fluorescence channel, configure PCR reaction reagents respectively and add the corresponding amplification templates to the reaction systems corresponding to each fluorescence channel, and conduct separate PCR amplification reaction experiments for each fluorescence channel;
[0014] S12: Observe the PCR amplification results of each fluorescence channel. When adding its corresponding amplification template to the first fluorescence channel for amplification, if it is observed that the second fluorescence channel also undergoes amplification with a Ct value close and a lower fluorescence increment ratio than the first fluorescence channel, it is determined that the first fluorescence channel provides fluorescence crosstalk to the second fluorescence channel, and the first fluorescence channel is determined as the fluorescence channel providing crosstalk, and the second fluorescence channel is determined as the fluorescence channel affected by crosstalk.
[0015] Preferably, for step S2, to conduct a PCR amplification reaction experiment to generate PCR amplification curves for the fluorescence channel providing crosstalk and the fluorescence channel affected by crosstalk respectively, specifically: conduct a separate PCR amplification reaction experiment for the fluorescence channel providing crosstalk, and collect the fluorescence data of the fluorescence channel providing crosstalk and the fluorescence channel affected by crosstalk respectively during the PCR amplification reaction experiment, both in the form of one-dimensional arrays. Use the fluorescence data of the fluorescence channel providing crosstalk as the abscissa and the fluorescence data of the fluorescence channel affected by crosstalk as the ordinate to draw the PCR amplification curve.
[0016] Preferably, for step S2, according to the generated PCR amplification curve, use the linear fitting method to calculate the crosstalk correction coefficient.
[0017] Preferably, for the case where the fluorescence channels providing crosstalk and the fluorescence channels affected by crosstalk are in a one-to-one correspondence, the specific steps of step S2 to step S4 are as follows:
[0018] S101: Select the fluorescence channel F1 providing crosstalk and the fluorescence channel F2 affected by crosstalk;
[0019] S102: Conduct a PCR amplification reaction experiment on the fluorescence channel F1 alone. During the reaction process, collect the fluorescence data A1 and A2 of the fluorescence channel F1 providing crosstalk and the fluorescence channel F2 affected by crosstalk respectively, and generate a PCR amplification curve L based on the obtained fluorescence data;
[0020] S103: Perform linear fitting using the PCR amplification curve L, calculate the obtained slope k, and use the slope k as the crosstalk correction coefficient between the two fluorescence channels;
[0021] S104: Conduct a PCR amplification reaction detection to obtain the fluorescence data A3 corresponding to the fluorescence channel F2 affected by crosstalk;
[0022] S105: According to the crosstalk correction coefficient k and the fluorescence data A3 of the fluorescence channel F2 affected by crosstalk, remove the crosstalk of the fluorescence channel F1 providing crosstalk from the obtained fluorescence data A3 to obtain the net fluorescence data A3' after crosstalk decoupling of the fluorescence channel F2 affected by crosstalk.
[0023] Preferably, the specific calculation formula of the linear fitting is as follows:
[0024] A2 i = k × A1 i + b, i = 1, 2,..., n,
[0025] where A1 i is the i-th fluorescence data of the fluorescence channel F1 providing crosstalk, A2 i is the i-th fluorescence data of the fluorescence channel F2 affected by crosstalk, k is the slope, i is the cycle number of the fluorescence data, n is the total number of fluorescence data, and b is a constant.
[0026] Preferably, for removing the crosstalk of the fluorescence channel F1 providing crosstalk from the obtained fluorescence data A3 in step S5 to obtain the net fluorescence data A3' after crosstalk decoupling of the fluorescence channel F2 affected by crosstalk, the specific calculation formula is as follows:
[0027] A3′ i = A3 i - k × A1 i , i = 1, 2,..., n
[0028] where A3 i ′ is the i-th fluorescence data of the fluorescence channel F2 after crosstalk decoupling, A3i The i-th fluorescence data of the fluorescence channel F2 affected by crosstalk, A1 i is the i-th fluorescence data of the fluorescence channel F1 that provides crosstalk, k is the slope, i is the cycle number of the fluorescence data, and n is the total number of the fluorescence data.
[0029] Preferably, for the case where there are multiple-to-one relationships between the fluorescence channels that provide crosstalk and the fluorescence channels affected by crosstalk, the specific steps of step S2 to step S4 are as follows:
[0030] S201: Select multiple fluorescence channels that provide crosstalk and the fluorescence channel F affected by crosstalk among the multiple fluorescence channels where fluorescence crosstalk occurs, where F j represents the j-th fluorescence channel that provides crosstalk, and m represents the number of fluorescence channels that provide crosstalk;
[0031] S202: Select a fluorescence channel F that provides crosstalk j , and only perform a PCR amplification reaction experiment on the fluorescence channel F that provides crosstalk j . During the reaction process, collect the fluorescence data of the fluorescence channel F that provides crosstalk j and the fluorescence channel F affected by crosstalk, and generate a PCR amplification curve L j ;
[0032] S203: Perform linear fitting using the PCR amplification curve L j to calculate the obtained slope k j , and use the slope k j as the crosstalk correction coefficient between the fluorescence channels F j and F;
[0033] S204: Repeat steps S202 to S203 to calculate the corresponding crosstalk correction coefficients for all fluorescence channels that provide crosstalk, and obtain the set of crosstalk correction coefficients
[0034] S205: Perform a PCR amplification reaction detection to obtain the fluorescence data of all fluorescence channels, including the fluorescence data of multiple fluorescence channels that provide crosstalk and the fluorescence data A of the fluorescence channel F affected by crosstalk;
[0035] S206: For the fluorescence channel F affected by crosstalk, according to the set of crosstalk correction coefficients and the fluorescence data of the fluorescence channel, remove the crosstalk of multiple fluorescence channels from the obtained fluorescence data A to obtain the net fluorescence data A' after crosstalk decoupling of the fluorescence channel F affected by crosstalk. The formula is
[0036] Compared with the prior art, the beneficial effects produced by the present invention are:
[0037] 1) The present invention directly calculates and generates a crosstalk correction coefficient based on the PCR amplification curves of the provided crosstalk and received crosstalk channels, and directly eliminates the crosstalk according to the crosstalk correction coefficient. Compared with the traditional method of calculating and correcting crosstalk through the excitation and emission spectrum ratios, the calculation principle of the method of the present invention is simple, the operation steps are simple, easy to implement, the correction effect is good, the accuracy of the obtained results is higher, the implementation cost is low, and it is suitable for wide promotion.
[0038] 2) For the complex case of multi-to-one crosstalk, the present invention can be split into multiple groups of one-to-one crosstalk. The correction coefficient is calculated for each group of crosstalk respectively, and then unified deduction is performed. The principle of the whole process is easy to understand, easy to implement, and the correction effect is accurate. BRIEF DESCRIPTION OF THE DRAWINGS
[0039] Figure 1 It is a flowchart of a method for decoupling fluorescence signal crosstalk in PCR detection according to an embodiment of the present invention;
[0040] Figure 2 It is a schematic diagram of the linear fitting result of fluorescence data for the one-to-one crosstalk case in Embodiment 3 of the present invention;
[0041] Figure 3 It is a schematic diagram of the comparison of fluorescence data before and after crosstalk decoupling for the one-to-one crosstalk case in Embodiment 3 of the present invention;
[0042] Figure 4 It is a schematic diagram of the result of linear fitting of fluorescence data for the two-to-one crosstalk case in Embodiment 4 of the present invention Figure 1 ;
[0043] Figure 5 It is a schematic diagram of the result of linear fitting of fluorescence data for the two-to-one crosstalk case in Embodiment 4 of the present invention Figure 2 ;
[0044] Figure 6 It is a schematic diagram of the comparison of fluorescence data before and after crosstalk decoupling for the two-to-one crosstalk case in Embodiment 4 of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS
[0045] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the Figures 1 to 6 , in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.
[0046] Embodiment 1
[0047] In combination with the attached Figure 1As shown in the figure, this embodiment provides a method for decoupling the fluorescence signal crosstalk in PCR detection, including the following steps:
[0048] S1: Determine the fluorescence channels where fluorescence crosstalk occurs, including the fluorescence channel that provides crosstalk and the fluorescence channel that is affected by crosstalk;
[0049] S2: Conduct a PCR amplification reaction experiment to generate PCR amplification curves for the fluorescence channel that provides crosstalk and the fluorescence channel that is affected by crosstalk respectively, and calculate the crosstalk correction coefficient between the two fluorescence channels according to the PCR amplification curves;
[0050] S3: Conduct a PCR amplification reaction detection to obtain the fluorescence data of each fluorescence channel;
[0051] S4: According to the crosstalk correction coefficient and the fluorescence data of each fluorescence channel, subtract the corresponding crosstalk of the fluorescence channel that provides crosstalk from the fluorescence data of the fluorescence channel that is affected by crosstalk, and the net fluorescence data after decoupling the fluorescence channel crosstalk can be obtained.
[0052] The present invention directly calculates and generates the crosstalk correction coefficient based on the PCR amplification curves of the two channels that provide crosstalk and receive crosstalk, and directly eliminates the crosstalk according to the crosstalk correction coefficient. Compared with the traditional method of calculating and correcting crosstalk through the excitation and emission spectrum ratios, the calculation principle of the method of the present invention is simple, the operation steps are simple, easy to implement, the correction effect is good, the accuracy of the obtained results is higher, the implementation cost is low, and it is suitable for wide promotion.
[0053] Example 2
[0054] Combined with the attached Figure 1 As shown in the figure, this embodiment is based on Example 1 and gives a further description of the implementation method of the specific content in the method. This embodiment provides a method for decoupling the fluorescence signal crosstalk in PCR detection, including the following steps:
[0055] S1: Determine the fluorescence channels where fluorescence crosstalk occurs, including the fluorescence channel that provides crosstalk and the fluorescence channel that is affected by crosstalk;
[0056] S2: Conduct a PCR amplification reaction experiment to generate PCR amplification curves for the fluorescence channel that provides crosstalk and the fluorescence channel that is affected by crosstalk respectively, and calculate the crosstalk correction coefficient between the two fluorescence channels according to the PCR amplification curves;
[0057] S3: Conduct a PCR amplification reaction detection to obtain the fluorescence data of each fluorescence channel;
[0058] S4: According to the crosstalk correction coefficient and the fluorescence data of each fluorescence channel, subtract the corresponding crosstalk of the fluorescence channel that provides crosstalk from the fluorescence data of the fluorescence channel that is affected by crosstalk, and the net fluorescence data after decoupling the fluorescence channel crosstalk can be obtained.
[0059] In this embodiment, in step S1, to determine the fluorescence channels where fluorescence crosstalk occurs, including the fluorescence channels providing crosstalk and the fluorescence channels affected by crosstalk, the following steps are included:
[0060] S11: For each fluorescence channel, configure PCR reaction reagents respectively and add the corresponding amplification templates to the reaction systems corresponding to each fluorescence channel, and conduct separate PCR amplification reaction experiments for each fluorescence channel;
[0061] S12: Observe the PCR amplification results of each fluorescence channel. For example, when adding its corresponding amplification template to the first fluorescence channel for amplification, if it is observed that the second fluorescence channel also has an amplification with a Ct value close and a fluorescence increment lower than that of the first fluorescence channel, it is determined that the first fluorescence channel provides fluorescence crosstalk to the second fluorescence channel, and the first fluorescence channel is determined as the fluorescence channel providing crosstalk, and the second fluorescence channel is determined as the fluorescence channel affected by crosstalk.
[0062] In this embodiment, in step S2, when conducting the PCR amplification reaction experiment, generate PCR amplification curves for the fluorescence channels providing crosstalk and the fluorescence channels affected by crosstalk respectively. Specifically: Conduct a separate PCR amplification reaction experiment for the fluorescence channel providing crosstalk, and during the PCR amplification reaction experiment, collect the fluorescence data of the fluorescence channels providing crosstalk and the fluorescence channels affected by crosstalk respectively, both in the form of one-dimensional arrays. Using the fluorescence data of the fluorescence channel providing crosstalk as the abscissa and the fluorescence data of the fluorescence channel affected by crosstalk as the ordinate, draw the PCR amplification curve.
[0063] In this embodiment, for step S2, according to the generated PCR amplification curve, a linear fitting method can be used to calculate the crosstalk correction coefficient. The calculation formula is: A2 i = k × A1 i + b, i = 1, 2,..., n, where A1 i is the i-th fluorescence data of the fluorescence channel F1 providing crosstalk, A2 i is the i-th fluorescence data of the fluorescence channel F2 affected by crosstalk, i is the cycle number of the fluorescence data, k is the slope, n is the total number of fluorescence data, n generally takes the value of 45, and b is a constant.
[0064] Embodiment 3
[0065] Combined with the attached Figures 2 - 3 As shown, this embodiment is based on Embodiment 2. For the one-to-one crosstalk situation (that is, the fluorescence channel providing crosstalk and the fluorescence channel affected by crosstalk are in a one-to-one situation), specific implementation steps for using the method of the present invention to achieve decoupling of PCR detection fluorescence signal crosstalk are specifically given, including the following steps:
[0066] S101: Select two fluorescence channels F1 and F2 that have fluorescence crosstalk, namely the fluorescence channel F1 that provides crosstalk and the fluorescence channel F2 that is affected by crosstalk.
[0067] S102: Conduct a PCR amplification reaction experiment on the fluorescence channel F1 alone. During the reaction process, collect the fluorescence data A1 and A2 of the fluorescence channel F1 that provides crosstalk and the fluorescence channel F2 that is affected by crosstalk respectively, and generate a PCR amplification curve L based on the obtained fluorescence data. For example, in a specific instance, the fluorescence data obtained from the two fluorescence channels are as follows:
[0068] A1 = [1468, 1473, 1481, 1488, 1484, 1484, 1485, 1482, 1481, 1486, 1470, 1479, 1470, 1484, 1485, 1456, 1463, 1473, 1460, 1468, 1462, 1474, 1456, 1452, 1465, 1480, 1468, 1466, 1452, 1474, 1481, 1478, 1484, 1493, 1544, 1606, 1658, 1771, 1843, 1937, 1965, 2033, 2092, 2135, 2164];
[0069] A2 = [1015, 1013, 983, 992, 1004, 997, 980, 998, 977, 982, 997, 1002, 998, 994, 1003, 1005, 1006, 991, 1000, 1016, 1015, 1022, 1033, 1015, 1012, 1007, 1006, 1006, 992, 1007, 1003, 1007, 1016, 1020, 1044, 1066, 1098, 1140, 1173, 1233, 1268, 1305, 1319, 1329, 1355].
[0070] S103: Perform linear fitting on the PCR amplification curve L (A2 i = k × A1 i + b, i = 1, 2,..., n), calculate the obtained slope k, and use the slope k as the crosstalk correction coefficient between the two fluorescence channels. In a specific instance, after performing linear fitting, we get A2 i = 0.5058 × A1 i + 257.87, (i = 1,..., 45), that is, the crosstalk slope k = 0.5058 (as Figure 2 shown).
[0071] S104: Perform a formal PCR amplification reaction test to obtain the fluorescence data A3 corresponding to the fluorescence channel F2. In a specific example, the fluorescence data A3 of the fluorescence channel F2 obtained from the PCR reaction is specifically:
[0072] A3 = [905, 893, 898, 900, 904, 914, 912, 915, 918, 911, 924, 937, 930, 936, 932, 934, 934, 939, 942, 938, 934, 939, 941, 942, 935, 933, 927, 949, 944, 939, 943, 941, 953, 950, 958, 992, 1005, 1043, 1059, 1108, 1121, 1144, 1156, 1191, 1209].
[0073] S105: According to the crosstalk correction coefficient k and the fluorescence data A3 of the fluorescence channel F2, remove the crosstalk of the fluorescence channel F1 from the obtained fluorescence data A3, and then the net fluorescence data A3' (A3′ i = A3 i - k × A1 i , i = 1, 2,..., n) can be obtained. In a specific example, let A3′ i = A3 i - 0.5058 × A1 i , i = 1, 2,..., 45. Finally, the net fluorescence value A3' of F2 is obtained, specifically:
[0074] A3' = [-30.642, -38.0898, -35.6188, -39.1826, -38.7232, -23.1594, -25.1594, -30.758, -28.7696, -31.2174, -19.7348, -2.6884, -10.1942, -8.2406, -8.1942, -11.2522, -8.2174, -7.2638, -2.7464, -3.2058, -13.2754, -2.7116, 0.3, 3.829, -15.816, -18.8276, -14.7116, -3.8392, -0.7464, -5.2406, -5.287, -9.816, 0.1608, -5.874, -19.1176, -3.3264, -16.1222, -5.4354, -14.2196, 3.9266, -12.9156, -10.1476, -19.3912, -10.6928, -0.7856].
[0075] Such as Figure 3As shown in the figure, by comparing A3 and A3', it can be found that after the crosstalk decoupling method of the present invention is applied, the false positive curve A3 caused by crosstalk has been transformed into a normal negative curve A3'. The present invention can effectively eliminate the crosstalk between fluorescence channels and improve the accuracy of PCR detection.
[0076] Example 4
[0077] Combined with the attached Figures 4 - 6 As shown in the figure, this embodiment is based on Example 2 and specifically gives the specific implementation steps for realizing the crosstalk decoupling of PCR detection fluorescence signals for the case of two-to-one crosstalk (that is, the fluorescence channels providing crosstalk and the fluorescence channels affected by crosstalk are in a two-to-one situation), including the following steps:
[0078] S201: Select three fluorescence channels F1, F2, and F3 where fluorescence crosstalk occurs, that is, the fluorescence channels F1 and F2 providing crosstalk, and the fluorescence channel F2 affected by crosstalk.
[0079] S202: Only conduct a PCR amplification reaction experiment on the fluorescence channel F1. During the reaction process, collect the fluorescence data A1 and A3 of the fluorescence channels F1 and F3 respectively, and generate a PCR amplification curve L1 based on the obtained fluorescence data. In the specific implementation process, only add the nucleic acid template of the fluorescence channel F1, so that only the fluorescence channel F1 undergoes PCR amplification, and collect the fluorescence data A1 and A3 of the fluorescence channels F1 and F3 respectively. In a specific example, the collected fluorescence data A1 and A3 are:
[0080] A1 = [2583, 2524, 2464, 2454, 2405, 2381, 2365, 2344, 2321, 2347, 2333, 2307, 2309, 2321, 2307, 2298, 2262, 2272, 2269, 2253, 2270, 2286, 2322, 2411, 2564, 2833, 3242, 3776, 4095, 4095, 4095, 4095, 4095, 4095, 4095, 4095, 4095, 4095, 4095, 4095, 4095, 4095, 4095];
[0081] A3 = [1026, 1023, 1006, 1015, 997, 1001, 1016, 1002, 993, 992, 989, 992, 993, 1006, 1001, 987, 985, 989, 976, 984, 988, 997, 1010, 1013, 1039, 1100, 1166, 1263, 1357, 1469, 1552, 1645, 1697, 1735, 1748, 1786, 1797, 1808, 1819, 1817, 1876, 1837, 1836, 1854, 1864, 1872].
[0082] S203: Use the PCR amplification curve L1 for linear fitting (A3 i = k1 × A1 i + b, i = 1, 2,..., n), calculate the obtained slope k1, which is the crosstalk correction coefficient between fluorescence channels F1 and F3. In a specific example, linear fitting is performed to obtain A3 i = 0.1915 × A1 i + 554.77, (i = 1,..., 45), that is, the crosstalk slope k1 = 0.1915 (as Figure 4 shown).
[0083] S204: Only perform PCR amplification reaction experiments on fluorescence channel F2. During the reaction process, collect the fluorescence data A2 and A4 of fluorescence channels F2 and F3 respectively, and generate a PCR amplification curve L2 based on the obtained fluorescence data. In the specific implementation process, only add the nucleic acid template of fluorescence channel F2, so that only fluorescence channel F2 undergoes PCR amplification, and collect the fluorescence data A2 and A4 of fluorescence channels F2 and F3 respectively. In a specific example, the collected fluorescence data A2 and A4 are:
[0084] A2 = [1054, 1066, 1070, 1069, 1067, 1067, 1083, 1070, 1059, 1075, 1063, 1099, 1074, 1067, 1066, 1072, 1080, 1086, 1083, 1088, 1114, 1139, 1183, 1296, 1455, 1674, 1998, 2316, 2648, 2927, 3205, 3378, 3504, 3621, 3693, 3727, 3759, 3789, 3814, 3804, 3857, 3852, 3886, 3877, 3899, 3908];
[0085] A4 = [1034, 1044, 1039, 1026, 1024, 1019, 1017, 1017, 1014, 1012, 1006, 1002, 1006, 1015, 1003, 1005, 1001, 1004, 1013, 999, 1012, 1013, 1013, 1016, 1046, 1073, 1104, 1137, 1188, 1231, 1241, 1268, 1276, 1291, 1300, 1315, 1295, 1312, 1325, 1327, 1322, 1321, 1322, 1318, 1319, 1323].
[0086] S205: Use the PCR amplification curve L2 for linear fitting (A4 i = k2 × A2 i + b, i = 1, 2,..., n), to obtain the slope k2, which is the crosstalk correction coefficient between fluorescence channels F2 and F3. In a specific example, linear fitting is performed to obtain A4 i = 0.1169 × A2 i + 872.5, (i = 1,..., 45), that is, the crosstalk slope k2 = 0.1169 (as Figure 5 shown).
[0087] S206: Conduct a formal PCR amplification reaction detection. The fluorescence data corresponding to the obtained fluorescence channels F1, F2, and F3 are A5, A6, and A7 respectively. In a specific example, the fluorescence data A5, A6, and A7 of the fluorescence channels F1, F2, and F3 obtained from the PCR reaction are specifically:
[0088] A5 = [866, 870, 865, 870, 875, 867, 870, 879, 868, 869, 877, 873, 874, 876, 879, 877, 882, 877, 887, 891, 922, 950, 1008, 1113, 1284, 1364, 1494, 1729, 1971, 2122, 2271, 2349, 2408, 2448, 2450, 2467, 2493, 2492, 2475, 2497, 2484, 2487, 2495, 2475, 2486, 2476];
[0089] A6 = [999, 991, 978, 972, 974, 966, 969, 976, 972, 977, 965, 979, 971, 956, 969, 951, 958, 978, 966, 968, 966, 982, 974, 997, 1011, 1061, 1120, 1198, 1287, 1370, 1408, 1468, 1521, 1527, 1572, 1587, 1595, 1611, 1609, 1616, 1638, 1643, 1640, 1642, 1633, 1640];
[0090] A7 = [2139, 2108, 2085, 2039, 2021, 1978, 1986, 1977, 1962, 1961, 1956, 1943, 1936, 1922, 1924, 1900, 1886, 1914, 1896, 1898, 1891, 1905, 1934, 1965, 2045, 2200, 2413, 2732, 3061, 3374, 3597, 3818, 3991, 4095, 4095, 4095, 4095, 4095, 4095, 4095, 4095, 4095, 4095, 4095, 4095];
[0091] S207: According to the crosstalk correction coefficients k1 and k2 and the fluorescence data A5, A6, and A7 of the fluorescence channels F1, F2, and F3, remove the crosstalk of the fluorescence channels F1 and F2 from the obtained fluorescence data A7, and the net fluorescence value of the fluorescence channel F3 can be obtained. The calculation formula is: A7 i ' = A7 i - k1 × A5 i - k2 × A6 i .
[0092] Let A7 i ' = A7 i - 0.1915 × A5 i - 0.1169 × A6 i , and finally obtain the net fluorescence value A3' of F6, specifically:
[0093] A7' = [615.9509, 623.5748, 621.2635, 631.6409, 638.7451, 635.7718, 637.8366, 647.8887, 638.6422, 639.7591, 648.3436, 645.8633, 647.6816, 651.3182, 654.0844, 654.89, 661.5266, 653.2534, 665.3576, 669.1238, 700.9421, 727.3055, 781.9154, 883.2915, 1044.9395, 1106.82, 1211.9203, 1409.6292, 1613.1691, 1727.5794, 1850.5107, 1902.6758, 1941.4521, 1969.2945, 1971.2945, 1988.2945, 2014.2945, 2013.2945, 1996.2945, 2018.2945, 2005.2945, 2008.2945, 2016.2945, 1996.2945, 2007.2945, 1997.2945].
[0094] As Figure 6 shown, by comparing A7 with A7', it can be found that after the processing of the crosstalk decoupling method of the present invention, the false positive curve A7 caused by crosstalk has been transformed into a normal negative curve A7'. The present invention can effectively eliminate the crosstalk between fluorescence channels and improve the accuracy of PCR detection.
[0095] The above are all preferred embodiments of the present invention, and the protection scope of the present invention is not limited accordingly. Therefore, all equivalent changes made according to the structure, shape, and principle of the present invention should be covered within the protection scope of the present invention.
Claims
1. A method for decoupling crosstalk of fluorescence signals in PCR detection, characterized in that, Including the following steps: S1: Determine the fluorescence channels with fluorescence crosstalk, including the fluorescence channel providing crosstalk and the fluorescence channel affected by crosstalk; S2: Conduct a PCR amplification reaction experiment, generate PCR amplification curves for the fluorescence channel providing crosstalk and the fluorescence channel affected by crosstalk respectively, and calculate the crosstalk correction coefficient between the two fluorescence channels according to the PCR amplification curves; S3: Conduct a PCR amplification reaction detection to obtain the fluorescence data of each fluorescence channel; S4: According to the crosstalk correction coefficient and the fluorescence data of each fluorescence channel, subtract the corresponding crosstalk of the fluorescence channel providing crosstalk from the fluorescence data of the fluorescence channel affected by crosstalk to obtain the net fluorescence data after decoupling the fluorescence channel crosstalk.
2. The method for decoupling the fluorescence signal crosstalk in PCR detection according to claim 1, wherein For step S1, to determine the fluorescence channels with fluorescence crosstalk, including the fluorescence channel providing crosstalk and the fluorescence channel affected by crosstalk, it includes the following steps: S11: For each fluorescence channel, configure PCR reaction reagents respectively and add the corresponding amplification template to the reaction system corresponding to each fluorescence channel, and conduct separate PCR amplification reaction experiments for each fluorescence channel; S12: Observe the PCR amplification results of each fluorescence channel. When adding its corresponding amplification template to the first fluorescence channel for amplification, if it is observed that the second fluorescence channel also undergoes amplification with a Ct value close and a fluorescence increment lower than that of the first fluorescence channel, it is determined that the first fluorescence channel provides fluorescence crosstalk to the second fluorescence channel, and the first fluorescence channel is determined as the fluorescence channel providing crosstalk, and the second fluorescence channel is determined as the fluorescence channel affected by crosstalk.
3. A method for decoupling fluorescence signal crosstalk in PCR detection according to claim 1, characterized in that For step S2, in the PCR amplification reaction experiment, generate PCR amplification curves for the fluorescence channel providing crosstalk and the fluorescence channel affected by crosstalk respectively. Specifically: Conduct a separate PCR amplification reaction experiment for the fluorescence channel providing crosstalk. During the PCR amplification reaction experiment, collect the fluorescence data of the fluorescence channel providing crosstalk and the fluorescence channel affected by crosstalk respectively, both in the form of one-dimensional arrays. Use the fluorescence data of the fluorescence channel providing crosstalk as the abscissa and the fluorescence data of the fluorescence channel affected by crosstalk as the ordinate to draw the PCR amplification curve.
4. A method for decoupling crosstalk of fluorescence signals in PCR detection according to claim 3, characterized in that, For step S2, according to the generated PCR amplification curve, use the linear fitting method to calculate the crosstalk correction coefficient.
5. A method for decoupling fluorescence signal crosstalk in PCR detection according to claim 4, characterized in that For the case where the fluorescence channel providing crosstalk and the fluorescence channel affected by crosstalk are one-to-one, the specific steps from step S2 to step S4 are: S101: Select the fluorescence channel F1 providing crosstalk and the fluorescence channel F2 affected by crosstalk; S102: Conduct a separate PCR amplification reaction experiment for the fluorescence channel F1. During the reaction process, collect the fluorescence data A1 and A2 of the fluorescence channel F1 providing crosstalk and the fluorescence channel F2 affected by crosstalk respectively, and generate a PCR amplification curve L based on the obtained fluorescence data; S103: Use the PCR amplification curve L for linear fitting, calculate the obtained slope k, and use the slope k as the crosstalk correction coefficient between the two fluorescence channels; S104: Conduct a PCR amplification reaction detection to obtain the fluorescence data A3 corresponding to the fluorescence channel F2 affected by crosstalk; S105: According to the crosstalk correction coefficient k and the fluorescence data A3 of the crosstalk-affected fluorescence channel F2, the crosstalk of the fluorescence channel F1 that provides crosstalk is removed from the obtained fluorescence data A3, and the net fluorescence data A3' after decoupling the crosstalk of the crosstalk-affected fluorescence channel F2 is obtained.
6. A method for decoupling fluorescence signal crosstalk in PCR detection according to claim 5, characterized in that The specific calculation formula of the linear fitting calculation formula is as follows: A2 i = k × A1 i + b, i = 1, 2,..., n, Among them, A1 i is the i-th fluorescence data of the fluorescence channel F1 that provides crosstalk, and A2 i is the i-th fluorescence data of the fluorescence channel F2 that is affected by crosstalk, k is the slope, i is the cycle number of the fluorescence data, n is the total number of fluorescence data, and b is a constant.
7. A method for decoupling crosstalk of PCR detection fluorescence signals according to claim 5, characterized in that For the removal of the crosstalk of the fluorescence channel F1 that provides crosstalk from the obtained fluorescence data A3 in step S5 to obtain the net fluorescence data A3' after decoupling the crosstalk of the crosstalk-affected fluorescence channel F2, the specific calculation formula is as follows: A3′ i = A3 i - k × A1 i , i = 1, 2, ..., n Among them, A3 i ′ is the i-th fluorescence data of the fluorescence channel F2 after crosstalk decoupling, and A3 i is the i-th fluorescence data of the fluorescence channel F2 affected by crosstalk. A1 i is the i-th fluorescence data of the fluorescence channel F1 providing crosstalk. k is the slope, i is the cycle number of the fluorescence data, and n is the total number of the fluorescence data.
8. A method for decoupling fluorescence signal crosstalk in PCR detection according to claim 4, characterized in that, For the case where there are multiple fluorescence channels providing crosstalk and one fluorescence channel being affected by crosstalk, the specific steps from step S2 to step S4 are as follows: S201: Select multiple fluorescence channels that provide crosstalk among multiple fluorescence channels where fluorescence crosstalk occurs and a fluorescence channel Fj that is subject to crosstalk, where Fj j represents the j-th fluorescence channel that provides crosstalk, and m represents the number of fluorescence channels that provide crosstalk; S202: Select a fluorescence channel F that provides crosstalk j , and only perform a PCR amplification reaction experiment on the fluorescence channel F that provides crosstalk j . During the reaction process, collect the fluorescence data of the fluorescence channel F that provides crosstalk j and the fluorescence channel F affected by crosstalk, and generate a PCR amplification curve L based on the obtained fluorescence data j ; S203: Utilize the PCR amplification curve L j to perform linear fitting and calculate the obtained slope k j , and use the slope k j as the crosstalk correction coefficient between the fluorescence channels F j and F; S204: Repeat steps S202 to S203 to calculate the corresponding crosstalk correction factors for all fluorescent channels that provide crosstalk, and obtain the set of crosstalk correction factors S205: Perform a PCR amplification reaction detection to obtain fluorescence data of all fluorescence channels, including the fluorescence data of multiple fluorescence channels that provide crosstalk and the fluorescence data A of the fluorescence channel F affected by crosstalk; S206: For the fluorescent channel F affected by crosstalk, according to the crosstalk correction coefficient set and the fluorescence data of the fluorescent channel, remove the crosstalk of multiple fluorescent channels from the obtained fluorescence data A to obtain the net fluorescence data A' after crosstalk decoupling of the fluorescent channel F affected by crosstalk. The formula is