Multi-PCR full-coverage primer design method and device

Abstract

The invention discloses a method and a device for designing a multiplex PCR (Polymerase Chain Reaction) full-coverage primer. The method comprises the following steps: designing a candidate primer pair set covering a whole target interval; calculating penalty scores of each group of candidate primer pairs and penalty scores when different candidate primer pair combinations coexist; calculating a penalty matrix of the primer combination and a coverage area matrix of the primers; and calculating an optimal primer combination by using integer programming. According to the technical scheme, the penalty score of each group of candidate primer pairs is calculated according to the extension degree of the primer dimer, and full coverage of an amplification region is ensured.

Description

Technical Field

[0001] The present invention relates to the technical field of PCR, and in particular, to a method and device for designing primers for full coverage of multiplex PCR. Background Art

[0002] Polymerase Chain Reaction (PCR for short) is a molecular biology technique used to amplify specific DNA fragments, and has a wide range of applications in the biological field. Multiplex PCR is an improvement based on conventional PCR, that is, more than two pairs of primers can be added to a reaction system to simultaneously amplify multiple nucleic acid fragments. Multiplex PCR technology can be applied in many aspects, including the detection of target microorganisms and the enrichment detection of drug-resistant genes. When amplifying genes, due to the actual read length limitation, if the gene length exceeds the read length, multiple pairs of overlapping primer pairs are required to achieve the purpose of full coverage sequencing of the target sequence. For example, when using a sequencing strategy of 150 bases at both ends (PE150), the length of the amplicon formed by the primer pair will be limited to within 300 bases. If the length of the target region is greater than 300 bases, then more than two amplicons are required to overlap each other to cover the entire region. Generally speaking, when there are multiple pairs of primer pairs in the system, it is necessary to consider the mutual interference between primers, such as primer dimers and non-specific amplification. The occurrence of primer dimers and non-specific amplification will cause a large amount of primers to be consumed and a lot of non-target sequences to be amplified, which is not conducive to the progress of the entire multiplex PCR amplification reaction. Therefore, while ensuring coverage of the target region, selecting primers with relatively good quality as much as possible and minimizing the mutual interference between primers are the keys to the design of a full coverage multiplex PCR system.

[0003] In this field, the general method for full coverage design of multiplex PCR is to use a greedy algorithm or a brute force algorithm. That is, after designing all candidate primers, the greedy algorithm will sort them according to certain rules, and then gradually put the primer pairs into the system until the entire target region is covered; the brute force algorithm directly enumerates all primer pair combinations that can cover the target region, and then selects the optimal system from them. The greedy algorithm is prone to situations where some parts cannot be covered, and the resulting system effect is not good. The brute force algorithm can theoretically perfectly obtain the best primer combination, but the design time is very long.

[0004] Therefore, a method for designing a multiplex PCR system that can obtain a relatively good one and ensure coverage of the entire target region within an acceptable design time is needed. Summary of the Invention

[0005] The present invention aims to provide a method and device for designing primers for multiplex PCR full coverage, so as to obtain a relatively good set of PCR primers that can cover the entire target region.

[0006] To achieve the above object, according to one aspect of the present invention, a method for designing multiplex PCR primers is provided. The method includes the following steps: designing a set of candidate primer pairs covering the entire target interval; calculating the penalty score of each group of candidate primer pairs and the penalty score when different combinations of candidate primer pairs coexist; calculating the penalty score matrix of the primer combination; calculating the coverage area matrix of the primers; and calculating the optimal primer combination using integer programming; wherein, calculating the penalty score of each group of candidate primer pairs and / or calculating the penalty score when different combinations of candidate primer pairs coexist includes calculating the primer dimer interference penalty score, and the penalty score when different combinations of candidate primer pairs coexist includes the penalty score of pairwise combinations of primers in different candidate primer pairs.

[0007] Further, calculating the primer dimer interference penalty score includes calculating the change in Gibbs free energy of the primer dimer formed by the forward primer and the reverse primer in the candidate primer pair or the pairwise combination of primers in different candidate primer pairs, and the primer dimer interference penalty score is calculated based on the degree of primer dimer extension determined according to the change in Gibbs free energy of the primer dimer.

[0008] Preferably, the number of candidate primer pairs is represented by n, and Pi and P j represent two pairs of candidate primers among them, i = 1, 2, 3,..., n, j = 1, 2, 3,..., n, i ≠ j; the change in Gibbs free energy of the primer dimer formed by the forward primer and the reverse primer in the candidate primer pair or the pairwise combination of primers in different candidate primer pairs is ΔG, and the primer dimer interference penalty score interactioni of the candidate primer pair or the primer dimer interference penalty score interactioni when different candidate primer pairs coexist j is obtained according to the following formula:

[0009]

[0010] Preferably, the primer dimer interference penalty score when different combinations of candidate primer pairs coexist includes selecting the maximum primer dimer interference penalty score among the pairwise combinations of primers as the final penalty score.

[0011] Further, calculating the penalty score of each group of candidate primer pairs or the penalty score when different combinations of candidate primer pairs coexist further includes one or more of the penalty score of the number of non-specific amplicons and the penalty score of nucleotide polymorphic sites on the primers;

[0012] Preferably, calculating the penalty score of each group of candidate primer pairs further includes the penalty score of the GC content of the primers;

[0013] Preferably, the number penalty for non-specific amplicons includes calculating the number penalty for non-specific amplicons in the background sequence;

[0014] Preferably, the background sequence includes other non-target sequences in the test sample except for the target sequence;

[0015] Preferably, the GC content penalty for primers includes giving different penalty weights according to different content ranges of the GC content;

[0016] More preferably, the GC content penalty for primers is calculated by the following formula:

[0017]

[0018] where gc_score i is the GC content penalty for the primer;

[0019] Preferably, the nucleotide polymorphism site penalty for a single primer is calculated by the following formula:

[0020] SNP i = 0.1 × the total number of SNPs on the primer + 1.2 × the number of SNPs in the 5 bases at the 3′ end of the primer. Preferably, when there is an SNP at the last base at the 3′ end of the primer, the primer pair is filtered out;

[0021] Preferably, the nucleotide polymorphism site penalty when candidate primer pairs or different combinations of candidate primer pairs coexist is equal to the sum of the nucleotide polymorphism site penalties of all primers within the candidate or combination;

[0022] Preferably, the penalty for each group of candidate primer pairs includes adding up the dimer interference penalty, the number penalty for non-specific amplicons, the GC content penalty for primers, and the nucleotide polymorphism site penalty on the primers; and / or

[0023] The penalty when different combinations of candidate primer pairs coexist includes adding up the total dimer interference penalty, the total number penalty for non-specific amplicons, and the total nucleotide polymorphism site penalty of the pairwise combinations of primers in different candidate primer pairs.

[0024] Furthermore, calculating the penalty matrix and the primer coverage region matrix for primer combinations includes recording the penalty when each group of candidate primer pairs or different combinations of candidate primer pairs coexist through the costM matrix, and calculating the primer coverage region matrix includes recording the region covered by the primers through the positionM matrix;

[0025] Preferably, the positionM matrix records the region covered by the primers, including recording the length of the region to be amplified as m, and initializing and generating an all-zero matrix positionM of m×n order; calculating the candidate primer pair P iCovered area S k (k = 1, 2, 3, ..., m), generate a 0-1 matrix;

[0026] Preferably, the costM matrix records the penalty scores when each group of the candidate primer pairs or different combinations of the candidate primer pairs coexist, including initializing and generating an all-zero matrix costM of order n×n; traversing all combinations of candidate primer pairs P i and P j (j < i), when i = j, replace the element in the i-th row and j-th column of the matrix with cost i ; when i ≠ j, replace the element in the i-th row and j-th column of the matrix with cost ij ;

[0027] Preferably, calculating the optimal primer combination using integer programming includes recording all combinations of primer pair selections as x, calculating the sum of costM and x, and solving the integer programming through constraint conditions. χ is the combination of primer pairs with the minimum sum of costM values after solving, and χ is the multiplex PCR primer combination;

[0028] More preferably, the constraint condition is that each column of positionM×x is an integer not less than 1 and not greater than 2.

[0029] According to another aspect of the present invention, there is provided an apparatus for designing multiplex PCR primers. The apparatus includes: a primer design module configured to design or input a set of candidate primer pairs covering the entire target interval; a penalty score calculation module including a penalty score unit configured to calculate the penalty score for each group of candidate primer pairs, a penalty score unit configured to calculate the penalty score when different combinations of candidate primer pairs coexist, a penalty matrix unit configured to calculate the penalty matrix of the primer combination, and a coverage area matrix unit configured to calculate the coverage area matrix of the primers; and an integer programming module configured to calculate the optimal primer combination using integer programming; wherein, the penalty score unit for calculating the penalty score for each group of candidate primer pairs or when different combinations of candidate primer pairs coexist is configured to include calculating the primer dimer interference penalty score, and the penalty score when different combinations of candidate primer pairs coexist includes the penalty scores of pairwise combinations of primers in different candidate primer pairs.

[0030] Further, calculating the primer dimer interference penalty score includes calculating the change in Gibbs free energy of the primer dimers formed by the forward and reverse primers in the candidate primer pairs or pairwise combinations of primers in different candidate primer pairs, and the primer dimer interference penalty score is calculated based on the degree of primer dimer extension determined according to the change in Gibbs free energy of the primer dimers;

[0031] Preferably, the number of candidate primer pairs is represented by n, and use P i and P jRepresenting two pairs of candidate primers among them, where i = 1, 2, 3, ..., n, j = 1, 2, 3, …, n, and i ≠ j; the Gibbs free energy change of the primer dimer formed by the forward primer and the reverse primer in the candidate primer pair or the pairwise combination of primers in different candidate primer pairs is ΔG, and the primer dimer interference penalty interactioni of the candidate primer pair or the primer dimer interference penalty interactioni when different candidate primer pairs coexist j including being obtained according to the following formula:

[0032]

[0033] Preferably, the primer dimer interference penalty when different candidate primer pairs coexist includes selecting the maximum primer dimer interference penalty among the pairwise combinations of primers as the final penalty.

[0034] Furthermore, the penalty calculation for each group of candidate primer pairs or the penalty when different candidate primer pairs coexist in the primer pair penalty unit is also set to include one or more of the number penalty of non-specific amplicons and the nucleotide polymorphism site penalty on the primer;

[0035] Preferably, the penalty calculation for each group of candidate primer pairs further includes the GC content penalty of the primer;

[0036] Preferably, the number penalty of non-specific amplicons includes calculating the number penalty of non-specific amplicons in the background sequence;

[0037] Preferably, the background sequence includes other non-target sequences in the test sample except the target sequence;

[0038] Preferably, the GC content penalty of the primer includes giving different penalty weights according to different content ranges of the GC content;

[0039] More preferably, the GC content penalty of the primer is obtained by calculating according to the following formula:

[0040]

[0041] where gc_score i is the GC content penalty of the primer;

[0042] Preferably, the nucleotide polymorphism site penalty on a single primer is obtained by calculating according to the following formula:

[0043] SNP i = 0.1 × the total number of SNPs on the primer + 1.2 × the number of SNPs in the 5 bases at the 3′ end of the primer

[0044] Preferably, when there is an SNP at the last base at the 3′ end of the primer, the primer pair is filtered out;

[0045] Preferably, when the candidate primer pairs or different combinations of the candidate primer pairs coexist, the nucleotide polymorphism site penalty is equal to the sum of the nucleotide polymorphism site penalties of all primers within the candidate or combination;

[0046] Preferably, the penalty score for each group of candidate primer pairs is obtained by adding up the dimer interference penalty score, the number penalty score of non-specific amplicons, the GC content penalty score of the primer, and the nucleotide polymorphism site penalty score of the primer; and / or

[0047] When different combinations of candidate primer pairs coexist, the penalty score is obtained by adding up the total penalty score of dimer interference penalties for pairwise combinations of primers in different candidate primer pairs, the total penalty score of the number penalty score of non-specific amplicons, and the total penalty score of nucleotide polymorphism site penalties.

[0048] Furthermore, the penalty score matrix for calculating the primer combination in the penalty score matrix unit is set to include recording the penalty score when each group of candidate primer pairs or different combinations of candidate primer pairs coexist through the costM matrix, and the coverage area matrix for calculating the primer in the coverage area matrix unit is set to include recording the area covered by the primer through the positionM matrix;

[0049] Preferably, the area covered by the primer recorded in the positionM matrix includes recording the length of the region to be amplified as m, and initializing to generate an all-zero matrix positionM of m×n order; calculating the region S covered by the candidate primer pair P i covered by k (k = 1, 2, 3,..., m), and generating a 0-1 matrix;

[0050] Preferably, the penalty score recorded in the costM matrix when each group of the candidate primer pairs or different combinations of the candidate primer pairs coexist includes initializing to generate an all-zero matrix costM of n×n order; traversing all combinations of candidate primer pairs P i and P j (j < i), when i = j, replacing the element in the i-th row and j-th column of the matrix with cost i ; when i ≠ j, replacing the element in the i-th row and j-th column of the matrix with cost ij ;

[0051] Preferably, the integer programming module includes recording all primer pair selection combinations as x, calculating the sum of costM and x, and performing integer programming solution through constraint conditions. χ is the primer pair selection combination with the smallest sum value of costM after solution, and χ is the multiplex PCR primer combination;

[0052] More preferably, the constraint condition is that each column of positionM×x is an integer not less than 1 and not greater than 2.

[0053] According to another aspect of the present invention, a computer-readable storage medium is provided. The storage medium includes a stored program, wherein when the program runs, it controls the device where the storage medium is located to execute the method for designing multiplex PCR primers of any one of the above.

[0054] According to another aspect of the present invention, a processor is provided. The processor is used to run a program, wherein when the program runs, it executes the method for designing multiplex PCR primers of any one of the above.

[0055] Applying the technical solution of the present invention, the penalty points of each group of candidate primer pairs are calculated according to the extension degree of primer dimers, allowing some primer dimers to exist, and ensuring full coverage of the amplification region to a certain extent; further, a multiplex PCR primer design scheme with the least interference between primers and satisfying full coverage of the target region is calculated by means of integer programming. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] The accompanying drawings forming a part of this application are used to provide a further understanding of the present invention. The schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation to the present invention. In the drawings:

[0057] Figure 1 shows a schematic flowchart of the design of multiplex PCR full-coverage primers according to an embodiment of the present invention;

[0058] Figure 2 shows the gel detection result diagram of the amplification product in Example 1;

[0059] Figure 3 shows the sequencing data depth result diagram of the UL27 interval amplified by the V1 primer set in Example 1; and

[0060] Figure 4 shows the sequencing data depth result diagram of the UL27 interval amplified by the V2 primer set in Example 1. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0061] It should be noted that, without conflict, the embodiments in this application and the features in the embodiments can be combined with each other. The present invention will be described in detail below with reference to the accompanying drawings and in combination with the embodiments.

[0062] Glossary:

[0063] Linear programming: Linear programming is a discipline that solves the optimal solution under certain constraints. By defining the objective optimization function and the corresponding constraints, the optimal solution is achieved by using mathematical methods.

[0064] Integer programming: Integer programming is a special type of linear programming. Generally, in linear programming, variables can be integers or fractions. However, for some specific problems, usually some variables must be integers. For example, when variables represent the number of machines or the number of workers. The linear programming with integer variables is called integer programming.

[0065] 0-1 programming: 0-1 programming is a type of integer programming and occupies an important position in integer programming. The variables in 0-1 programming are specified to take values between 0 and 1, which are bounded integer variables. Many practical problems such as assignment problems and site selection problems can be reduced to this type of programming, and primer design can also be reduced to this type of programming.

[0066] Non-specific amplification of primers: Non-specific amplification of primers refers to the situation where the current primer combination can produce an amplification effect in the template sequence or background sequence, but not at the position set by the designer. All amplification effects that occur at non-expected positions are called non-specific amplifications. Non-specific amplifications between primers may lead to the enrichment of non-target interval sequences and also consume the primers in the reaction pool.

[0067] Primer dimer: A primer dimer is formed when the bases at the 3' end of a primer itself hybridize with itself or other primers and extend at the 3' end under the action of Taq enzyme to form an amplification product. The interaction between these primers will competitively inhibit the binding to the target DNA, resulting in a decrease in amplification efficiency and primer yield. Primer dimers can also be divided into three types according to whether the two 3' ends of primer binding can be extended: both ends can be extended, only one end can be extended, and cannot be extended.

[0068] Single Nucleotide Polymorphism (SNP) of primers: A single nucleotide polymorphism refers to a variation found in the genome where a specific base is replaced by another base. The presence of SNPs on primers will cause primer mismatches with the sequence when the target sequence has this mutation, resulting in a decrease in primer amplification efficiency or even inability to amplify.

[0069] Change in Gibbs free energy ΔG: Gibbs free energy is a thermodynamic function introduced in chemical thermodynamics to judge the direction of a process. In a closed system under isothermal and isobaric conditions, any spontaneous reaction proceeds in the direction of decreasing Gibbs free energy. Therefore, when the change in Gibbs free energy is negative, the reaction can proceed spontaneously.

[0070] In order to obtain a relatively good PCR primer set that can cover the entire target region, the present invention proposes the following technical solutions. See Figure 1 。

[0071] According to a typical embodiment of the present invention, a method for designing multiplex PCR primers is provided. The method includes the following steps: designing or inputting a set of candidate primer pairs covering the entire target region; calculating the penalty points for each group of candidate primer pairs and calculating the penalty points when different combinations of candidate primer pairs coexist; calculating the penalty matrix of primer combinations; calculating the coverage area matrix of primers; and calculating the optimal primer combination using integer programming; wherein, calculating the penalty points for each group of candidate primer pairs and / or calculating the penalty points when different combinations of candidate primer pairs coexist includes calculating the primer dimer interference penalty points, and the penalty points when different combinations of candidate primer pairs coexist include the penalty points for pairwise combinations of primers in different candidate primer pairs.

[0072] Applying the technical solution of the present invention, the penalty points for each group of candidate primer pairs are calculated according to the extension degree of primer dimers, allowing partial primer dimers to exist, and ensuring full coverage of the amplification region to a certain extent; further, an optimal multiplex PCR primer design scheme with the least interference between primers and full coverage of the target region is calculated by means of integer programming.

[0073] In an embodiment of the present invention, calculating the primer dimer interference penalty points includes calculating the change in Gibbs free energy of the primer dimer formed by the forward primer and the reverse primer in the candidate primer pair or pairwise combinations of primers in different candidate primer pairs, and the primer dimer interference penalty points are calculated based on the extension degree of the primer dimer determined according to the change in Gibbs free energy of the primer dimer.

[0074] Preferably, the number of candidate primer pairs is represented by n, and P i and P j represent two pairs of candidate primers among them, i = 1, 2, 3,..., n, j = 1, 2, 3,..., n, i ≠ j; the change in Gibbs free energy of the primer dimer formed by the forward primer and the reverse primer in the candidate primer pair or pairwise combinations of primers in different candidate primer pairs is ΔG, and the primer dimer interference penalty points interaction i or the primer dimer interference penalty points interaction ij when different said candidate primer pairs coexist are obtained according to the following formula:

[0075]

[0076] Preferably, the primer dimer interference penalty points when different combinations of candidate primer pairs coexist include selecting the maximum primer dimer interference penalty points among the pairwise combinations of primers as the final penalty points.

[0077] According to a typical embodiment of the present invention, calculating the penalty score for each group of candidate primer pairs or the penalty score when different combinations of candidate primer pairs coexist further includes one or more of the penalty score for the number of non-specific amplicons and the penalty score for nucleotide polymorphic sites on the primers; preferably, calculating the penalty score for each group of candidate primer pairs further includes the penalty score for the GC content of the primers; in order to more comprehensively and accurately evaluate the candidate primer pairs, preferably, the penalty score for each group of candidate primer pairs is obtained by adding up the penalty score for the number of non-specific amplicons, the penalty score for the GC content of the primers, and the penalty score for nucleotide polymorphic sites on the primers.

[0078] In the present invention, preferably, the penalty score for the number of non-specific amplicons includes calculating the penalty score for the number of non-specific amplicons in the background sequence. Preferably, the background sequence includes other non-target sequences in the test sample except the target sequence. Calculate the GC content of the amplicon formed by primer pair P i Because too high GC content of the amplicon will lead to poor amplification efficiency. Preferably, the penalty score for the GC content of the primers is given different penalty score weights according to different content ranges of the GC content, so as to more accurately score the GC content of the primers; for example, the penalty score for the GC content of the primers is calculated according to the following formula:

[0079]

[0080] wherein, gc_score i is the penalty score for the GC content of the primers.

[0081] According to a typical embodiment of the present invention, preferably, the penalty score for nucleotide polymorphic sites on a single primer includes

[0082] is calculated by the following formula:

[0083] SNP i = 0.1 × the total number of SNPs on the primer + 1.2 × the number of SNPs in the 5 bases at the 3′ end of the primer

[0084] Preferably, when there is an SNP at the last base at the 3′ end of the primer, filter out this primer pair;

[0085] Preferably, the penalty score for nucleotide polymorphic sites when the candidate primer pairs or different combinations of the candidate primer pairs coexist is equal to the sum of the penalty scores for nucleotide polymorphic sites of all primers within the candidate or combination.

[0086] Preferably, the penalty score of each group of candidate primer pairs is obtained by adding up the dimer interference penalty score, the number penalty score of non-specific amplicons, the GC content penalty score of the primers, and the nucleotide polymorphism site penalty score on the primers; and / or the penalty score when different combinations of candidate primer pairs coexist is obtained by adding up the total penalty score of dimer interference penalties for pairwise combinations of primers in different candidate primer pairs, the total penalty score of the number penalty score of non-specific amplicons, and the total penalty score of nucleotide polymorphism site penalties.

[0087] According to a typical embodiment of the present invention, calculating the penalty score when different combinations of candidate primer pairs coexist includes: calculating the Gibbs free energy ΔG of the primer dimers formed by the four primers with each other, and calculating the dimer interference penalty score, and taking the maximum value as the final total dimer interference penalty score; determining the theoretically binding regions of the four primers on the target sequence and the background sequence and the formed theoretical amplicons, comparing the theoretical amplicons with the set amplicons, if the theoretical amplicons are not the set amplicons, then the theoretical amplicons are non-specific amplifications and are included in the number penalty score of non-specific amplicons, and finally obtaining the total number penalty score of non-specific amplicons; calculating the nucleotide polymorphism site penalty score on each of the four primers, and adding up the nucleotide polymorphism site penalty scores of all single primers to obtain the total nucleotide polymorphism site penalty score; adding up the total dimer interference penalty score, the total number penalty score of non-specific amplicons, and the total nucleotide polymorphism site penalty score to obtain the penalty score when different combinations of candidate primer pairs coexist. Such a setting can more comprehensively and accurately reflect the situation when different combinations of candidate primer pairs coexist.

[0088] According to a typical embodiment of the present invention, calculating the penalty score matrix and the primer coverage region matrix of the primer combinations includes recording the penalty scores between each group of candidate primer pairs or when different combinations of the candidate primer pairs coexist through the costM matrix, and calculating the primer coverage region matrix includes recording the regions covered by the primers through the positionM matrix; preferably, the positionM matrix record includes recording the length of the region to be amplified as m, and preferably, initializing and generating an all-zero matrix positionM of m×n order; calculating the region S covered by the candidate primer pair P i covered by k (k = 1, 2, 3,..., m), replacing the 0 in the i-th row and k-th column of the matrix with 1, representing that the i-th primer covers the j-th base on the target sequence, and generating a 0-1 matrix; the costM matrix records the penalty scores between each group of the candidate primer pairs or when different combinations of the candidate primer pairs coexist, including initializing and generating an all-zero matrix costM of n×n order; traversing all combinations of candidate primer pairs Pi and P j(j < i; i, j = 1, 2, 3,..., n), when i = j, replace the element in the i-th row and j-th column of the matrix with costi; when i ≠ j, replace the element in the i-th row and j-th column of the matrix with costi j ; Preferably, calculating the optimal primer combination using integer programming includes recording all primer pair selection combinations as x, where x is an n×1 0-1 matrix. When primer pair P i is selected, the i-th column in the matrix is replaced with 1, otherwise it is 0; record the optimal combination as χ, and the sum of costM×χ is the smallest among all combinations; through the constraint conditions, in the way of integer programming, calculate χ from x, and χ is the multiplex PCR primer combination; more preferably, the constraint condition is that each column of positionM×x is an integer not less than 1 and not greater than 2; to ensure that each position on the gene is covered by the primer set and there is no excessive duplication.

[0089] According to another aspect of the present invention, there is provided an apparatus for designing multiplex PCR primers. The apparatus includes: a primer design module configured to design or input a set of candidate primer pairs covering the entire target region; a penalty score calculation module including a penalty score unit configured to calculate the penalty score of each group of candidate primer pairs, a penalty score unit configured to calculate the penalty score when different candidate primer pair combinations coexist, a penalty score matrix unit configured to calculate the penalty score matrix of the primer combination, and a coverage region matrix unit configured to calculate the coverage region matrix of the primers; and an integer programming module configured to calculate the optimal primer combination using integer programming; wherein, the penalty score calculation for each group of candidate primer pairs or when different candidate primer pair combinations coexist in the primer pair penalty score unit is set to include calculating the primer dimer interference penalty score, and the penalty score when different candidate primer pair combinations coexist includes the penalty scores of pairwise combinations of primers in different candidate primer pairs.

[0090] Applying the technical solution of the present invention, calculate the penalty score of each group of candidate primer pairs according to the primer dimer extension degree, allow some primer dimers to exist, and ensure full coverage of the amplification region to a certain extent; further calculate the multiplex PCR primer design scheme with the least interference between primers and satisfying full coverage of the target region through integer programming.

[0091] In an embodiment of the present invention, calculating the primer dimer interference penalty score includes calculating the change in Gibbs free energy of the primer dimer formed by the forward primer and the reverse primer in the candidate primer pair or pairwise combinations of primers in different candidate primer pairs, and the primer dimer interference penalty score is calculated based on the primer dimer extension degree determined by the change in Gibbs free energy of the primer dimer;

[0092] Preferably, the number of candidate primer pairs is represented by n, and use P i and P jRepresenting two pairs of candidate primers among them, where i = 1, 2, 3, ..., n, j = 1, 2, 3, …, n, and i ≠ j; the Gibbs free energy change of the primer dimer formed by the forward primer and the reverse primer in the candidate primer pair or the pairwise combination of primers in different candidate primer pairs is ΔG, and the primer dimer interference penalty score interaction i or the primer dimer interference penalty score interaction when different said candidate primer pairs coexist ij is obtained according to the following formula:

[0093]

[0094] Preferably, the primer dimer interference penalty score when different candidate primer pairs coexist includes selecting the maximum primer dimer interference penalty score among the pairwise combinations of primers as the final penalty score.

[0095] According to a typical embodiment of the present invention, the penalty score calculation for each group of candidate primer pairs or the penalty score when different candidate primer pairs coexist in the primer pair penalty unit is also set to include one or more of the number penalty score of non-specific amplicons and the nucleotide polymorphism site penalty score on the primer; preferably, the penalty score calculation for each group of candidate primer pairs also includes the GC content penalty score of the primer; in order to more comprehensively and accurately evaluate the candidate primer pairs, preferably, the penalty score for each group of candidate primer pairs is obtained by adding the number penalty score of non-specific amplicons, the GC content penalty score of the primer, and the nucleotide polymorphism site penalty score on the primer.

[0096] In the present invention, preferably, the number penalty score of non-specific amplicons includes calculating the number penalty score of non-specific amplicons in the background sequence; preferably, the background sequence includes other non-target sequences in the detection sample except the target sequence; preferably, the GC content penalty score of the primer includes giving different penalty weights according to different GC content ranges; more preferably, the GC content penalty score of the primer is calculated according to the following formula:

[0097]

[0098] where gc_score i is the GC content penalty score of the primer.

[0099] According to a typical embodiment of the present invention, preferably, the nucleotide polymorphism site penalty score of a single primer is calculated according to the following formula:

[0100] SNP i = 0.1 × the total number of SNPs on the primer + 1.2 × the number of SNPs in the 5 bases at the 3′ end of the primer

[0101] Preferably, when there is a SNP at the last base at the 3' end of the primer, the primer pair is filtered out;

[0102] Preferably, the nucleotide polymorphic site penalty on the primer pair is equal to the sum of the nucleotide polymorphic site penalties of all primers;

[0103] Preferably, the penalty for each group of candidate primer pairs includes the sum of the dimer interference penalty, the number penalty of non-specific amplicons, the GC content penalty of the primers, and the nucleotide polymorphic site penalty on the primers; and / or the penalty for different candidate primer pairs coexisting in combination includes the sum of the total dimer interference penalty, the total penalty for the number of non-specific amplicons, and the total penalty for the nucleotide polymorphic site penalty of the primers in different candidate primer pairs.

[0104] According to a typical embodiment of the present invention, the penalty matrix for calculating the primer combination in the penalty matrix unit is set to include recording the penalty between each group of candidate primer pairs or when different candidate primer pairs coexist through the costM matrix, and the coverage area matrix for calculating the primers in the coverage area matrix unit is set to include recording the area covered by the primers through the positionM matrix; preferably, the positionM matrix records the area covered by the primers, including recording the length of the region to be amplified as m, and initializing to generate an m×n-order all-zero matrix positionM; calculating the candidate primer pair P i Covered area S k (k=1, 2, 3, ..., m), generating a 0-1 matrix; preferably, the costM matrix records the penalty between each group of the candidate primer pairs or when different combinations of the candidate primer pairs coexist, including initializing and generating an n×n-order all-zero matrix costM; traversing all candidate primer pair combinations Pi and P j (j <i;i,j=1,2,3,...,n),当i=j时,将矩阵中的第i行第j列替换为cost i ; When i≠j, replace the i-th row and j-th column in the matrix with costi j ; Preferably, the integer programming module includes recording all primer pair selection combinations as x, calculating the sum of costM and x, and solving the integer programming through constraints, where χ is the primer pair selection combination with the smallest costM sum after solution, and χ is the multiple PCR primer combination; more preferably, the constraint condition is that each column of positionM×x is an integer not less than 1 and not greater than 2.

[0105] According to another aspect of the present invention, a computer-readable storage medium is provided, wherein the storage medium includes a stored program, wherein when the program is executed, the device where the storage medium is located is controlled to execute any of the above-mentioned multiplex PCR primer design methods.

[0106] According to another aspect of the present invention, a processor is provided. The processor is used to run a program, and when the program runs, it executes the method for multiplex PCR primer design described in any one of the above.

[0107] Optionally, the above electronic device may further include a transmission device and an input / output device, wherein the transmission device is connected to the above processor, and the input / output device is connected to the above processor.

[0108] Optionally, the specific examples in this embodiment may refer to the examples described in the above embodiments and optional implementation manners, and will not be elaborated herein.

[0109] Obviously, those skilled in the art should understand that the above modules or steps of the present invention can be implemented by a general-purpose computing device. They can be concentrated on a single computing device or distributed on a network composed of multiple computing devices. Optionally, they can be implemented by program codes executable by the computing device, so that they can be stored in a storage device and executed by the computing device. And in some cases, the steps shown or described can be executed in a different order from here, or they can be separately made into individual integrated circuit modules, or multiple modules or steps among them can be made into a single integrated circuit module to implement. In this way, the present invention is not limited to any specific combination of hardware and software.

[0110] The beneficial effects of the present invention will be further described below in conjunction with embodiments.

[0111] Embodiment 1

[0112] I. Materials

[0113] The sample template in this example is derived from the UL27 gene sequence in human cytomegalovirus (CMV), that is, from the 33183rd base to the 35009th base in the CMV reference genome NC_006273.2.

[0114] II. Methods

[0115] The specific operation process is as follows:

[0116] (1) Input the sequence of the region to be amplified, and use the primer3 software to uniformly design all candidate primers. Ensure that all candidate primers can cover the target region. The number of candidate primer pairs is represented by n, and P i represents a certain pair of candidate primers (i = 1, 2, 3,..., n).

[0117] (2) Calculate the penalty score of each group of candidate primer pairs. The higher the penalty score, the worse the amplification effect of the primer.

[0118] S1: Calculate the primer pair P iThe Gibbs free energy change ΔG (unit: kcal / mol) of the primer dimer formed by the forward primer and the reverse primer in

[0119]

[0120] S2: Calculate the number penalty of non-specific amplicons of primer pair P i in the background sequence, denoted as nonsp i .

[0121] S3: Calculate the GC content of the amplicon formed by primer pair P i . Since too high GC content of the amplicon will lead to poor amplification efficiency, different penalty weights are given according to different stages of the GC content. The penalty score of the GC content (gc_score i ) can be calculated according to the following formula:

[0122]

[0123] S4: Calculate the number of SNPs on each primer in primer pair P i . When there is an SNP at the last base at the 3' end of the primer, this pair of primer pairs needs to be filtered out. The SNP penalty of a single primer is as follows, and the SNP i penalty of the primer pair is equal to the sum of the SNP penalties of all primers.

[0124] SNP i = 0.1 × the total number of SNPs on the primer + 1.2 × the number of SNPs in the 5 bases at the 3' end of the primer

[0125] S5: The penalty score of each primer pair P i is obtained by summing the penalties of the above four parts:

[0126] cost i = interaction i + nonsp i + gc_score i + SNP i

[0127] (3) Calculate the penalty scores when different primer pair combinations Pi and P j (j ≠ i, j = 1, 2, 3,..., n) coexist. The higher the penalty score, the less compatible these two primer pairs are.

[0128] S1: Calculate the Gibbs free energy ΔG of the primer dimers formed by the four primers with each other, and calculate the dimer interference penalty score interactionij , take the maximum value among them as the finally obtained total penalty score.

[0129] S2: Determine the regions where the four primers theoretically bind to the target sequence and the background sequence, as well as the theoretically formed amplicons, and compare them with the set amplicons. If the theoretical amplicon is not the set amplicon, then this theoretical amplicon is a non-specific amplification and is counted as the non-specific amplification number penalty nonsp i .

[0130] The background sequence represents other non-target sequences in the test sample. For example, when detecting human pathogenic microorganisms, the background sequence can be the human genome sequence.

[0131] S3: Calculate the SNP penalty for each of the four primers, and finally sum up all the SNP penalties of the single primers to obtain the total penalty score SNPi j .

[0132] S4: The penalty for the primer pair combination Pi and P j is obtained by summing up the above penalties:

[0133] cost ij = interaction ij + nonsp ij + SNP ij

[0134] (4) Denote the length of the region to be amplified as m, and initialize to generate an all-zero matrix positionM of order m×n; calculate the region S i covered by the primer pair P k (k = 1, 2, 3,..., m), replace the 0 in the k-th column of the i-th row in the matrix with 1, indicating that the i-th primer covers the j-th base on the target sequence, and generate a 0-1 matrix.

[0135]

[0136] (5) Initialize to generate an all-zero matrix costM of order n×n; traverse all candidate primer pair combinations Pi and P j (j < i; i, j = 1, 2, 3,..., n), when i = j, replace the i-th row and j-th column in the matrix with cost i ; when i ≠ j, replace the i-th row and j-th column in the matrix with cost ij .

[0137]

[0138] (6) Denote all the primer pair selection combinations as x. x is a 0-1 matrix of order n×1. When the primer pair Pi When selected, the i-th column in the matrix will be replaced by 1, otherwise it is 0. Denote the optimal combination among them as χ, and the sum of costM×χ should be the smallest among all combinations. Through the following constraint conditions, in the way of integer programming, χ is calculated from x, and χ is the multiplex PCR primer combination with the least interference between primers and ensuring full coverage.

[0139] By formulating the constraint condition: each column of positionM×x is an integer not less than 1 and not greater than 2, the primer combination that can fully cover the UL27 gene sequence is calculated.

[0140] Table 1 Primer set V1 designed by the greedy algorithm

[0141]

[0142] Table 2 Primer set V2 designed by the algorithm of this embodiment

[0143]

[0144] III. Experimental verification

[0145] (1) Materials

[0146] PCR reaction system: nuclease-free water ddH2O, Ax DNAPolymerase (Guangdong BioPerfectus Technologies Co., Ltd., 5U / μL), Ax Reaction buffer (Guangdong BioPerfectus Technologies Co., Ltd.), the synthesized sequence of the CMV virus UL27 targeting region; primer set V1 designed by the greedy algorithm process and primer set V2 designed by this embodiment, 40 nM for each primer;

[0147] Instruments: PCR instrument, pipettor, micro high-speed centrifuge, shaker mixer, ultraviolet gel imaging analysis system (for agarose gel electrophoresis analysis);

[0148] Reagents: agarose, 1xTAE solution, low-toxic and high-sensitivity dye (recommended for pre-electrophoresis staining / gel staining method, for agarose gel electrophoresis), DL2,000 DNA Maker, nuclease-free water ddH2O;

[0149] Consumables: sterile 0.2 ml centrifuge tubes, sterile pipette tips of various models (matched with pipettors), marker pens.

[0150] (2) Methods

[0151] 1. Prepare 3 multiplex PCR reaction systems. In 4 sterile 0.2 mL centrifuge tubes (numbered ①, ②, ③, and ④ respectively), add nuclease-free water ddH2O, Ax DNA Polymerase (Guangdong BioPerfectus Technologies Co., Ltd., 5 U / μL), and Ax Reaction buffer (Guangdong BioPerfectus Technologies Co., Ltd.) simultaneously and separately.

[0152] 2. In tube ① (system negative control), add V1 primer mix and clinical sample (positive template).

[0153] In tube ②, add V1 primer mix and nuclease-free water ddH2O (negative template).

[0154] 3. In tube ③ (system negative control), add V2 primer mix and clinical sample (positive template).

[0155] In tube ④, add V2 primer mix and nuclease-free water ddH2O (negative template).

[0156] 3. After preparation, vortex and mix well, briefly centrifuge, and then run the amplification program in the same PCR instrument.

[0157] 4. After completion, perform 3% agarose gel electrophoresis analysis with a high-sensitivity dye (select DL2,000 DNA Maker as the amplification product size control during electrophoresis).

[0158] Experimental results: As Figure 2 shown, the target band is between 250 - 500 bp, and the brightness of the amplification band in tube ③ is greater than that in tube ①, indicating that the amplification efficiency of the V2 primer group mix is higher than that of the V1 primer group mix.

[0159] 5. Library construction and sequencing

[0160] 5.1. Product purification:

[0161] 1) Add 50 μL of magnetic beads to the reaction product, shake and mix well, and centrifuge.

[0162] 2) Let it stand at room temperature for 5 min, place it on the magnetic rack, and let it stand for 3 min.

[0163] 3) After the supernatant becomes clear, discard the supernatant.

[0164] 4) Wash the magnetic beads with 200 μL of 80% ethanol several times.

[0165] 5) Add TE for elution.

[0166] 5.2. End repair and addition of "A" tail:

[0167] 1) Prepare the system according to the following description:

[0168]

[0169] 2) Magnetic bead purification:

[0170] ① Add 50 μL of magnetic beads to the above reaction solution, mix well by shaking and centrifuge; ② Let it stand at room temperature for 5 min, place it on a magnetic stand and let it stand for 3 min; ③ After the supernatant is clear, discard the supernatant;

[0171] ④ Wash the magnetic beads with 200 μL of 80% ethanol for several times;

[0172] ⑤ Add TE for elution.

[0173] 5.3. Adapter ligation:

[0174] 1) Prepare the adapter amplification premix according to the following description:

[0175]

[0176] 2) Magnetic bead purification:

[0177] ① Add 50 μL of magnetic beads to the above reaction solution, mix well by shaking and centrifuge; ② Let it stand at room temperature for 5 min, place it on a magnetic stand and let it stand for 3 min; ③ After the supernatant is clear, discard the supernatant;

[0178] ④ Wash the magnetic beads with 200 μL of 80% ethanol for several times;

[0179] ⑤ Add TE for elution.

[0180] 6. PCR amplification

[0181] 1) Prepare the PCR amplification system according to the following description:

[0182]

[0183] 2) Product purification:

[0184] ① Add 50 μL of magnetic beads to the above reaction solution, and mix well by repeatedly pipetting;

[0185] ② Let it stand at room temperature for 5 min, place it on a magnetic stand and let it stand for 3 min;

[0186] ③ After the supernatant is clear, discard the supernatant;

[0187] ④ Wash the magnetic beads with 200 μL of 80% ethanol for several times;

[0188] ⑤ Resuspend the magnetic beads with TE and let it stand at room temperature for 1 min;

[0189] ⑥ Pipette 20 μL of the supernatant for library identification.

[0190] 2) Product purification:

[0191] ① Add 50 μL of magnetic beads to the above reaction solution, and use a pipette to aspirate and blow repeatedly to mix evenly;

[0192] ② Let it stand at room temperature for 5 min, place it on a magnetic stand, and let it stand for 3 min;

[0193] ③ After the supernatant is clarified, remove the supernatant;

[0194] ④ Wash the magnetic beads with 200 μL of 80% ethanol and repeat several times;

[0195] ⑤ Add TE to resuspend the magnetic beads and let it stand at room temperature for 1 min;

[0196] ⑥ Aspirate 20 μL of the supernatant for library identification.

[0197] 7. Sequencing

[0198] Send the purified library for sequencing, and align the sequenced data after sequencing to the reference genome using alignment software. Calculate the depth covered at each position (to make the differences easier to see, the depth is limited to less than 1000): Figure 3 The depth results of the sequencing data of the UL27 region amplified by the V1 primer set are shown; Figure 4 The depth results of the sequencing data of the UL27 region amplified by the V2 primer set are shown.

[0199] IV. Results

[0200] In this example, after designing primers for the UL27 region using the above algorithm method, from Figure 3 and Figure 4 the sequencing results, it can be seen that the coverage of the V2 primer set of this algorithm is significantly higher than that of the V1 primer set designed by the greedy algorithm, and the amplification efficiency at some positions (such as 33769 - 33823, 34115 - 34184, 34522 - 34705) is also better than that of the primers designed by the greedy algorithm.

[0201] From the above description, it can be seen that the above embodiments of the present invention achieve the following technical effects: The quality of the primer combination is measured by the non-specific amplification, primer dimers, single nucleotide polymorphism (SNP) on the primer, and the GC content of the primer generated between the primers. Constraint conditions are set according to the condition of full coverage, and the primer pool is designed using integer programming. Finally, a multiplex PCR primer system with less interference between primers and ensuring full coverage is obtained.

[0202] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A method for designing multiplex PCR primers, characterized in that, Comprising the following steps: Designing or inputting a set of candidate primer pairs covering the entire targeted interval; Calculating the penalty scores of each group of candidate primer pairs and calculating the penalty scores when different combinations of candidate primer pairs coexist; Calculating the penalty score matrix of primer combinations; Calculating the coverage area matrix of primers; And Calculating the optimal primer combination using integer programming; Wherein, calculating the penalty score of each group of the candidate primer pairs and / or calculating the penalty score when different combinations of the candidate primer pairs coexist includes calculating the primer dimer interference penalty score, and the penalty score when the different combinations of the candidate primer pairs coexist includes the penalty scores of pairwise combinations of primers in the different candidate primer pairs.

2. The method according to claim 1, wherein Calculating the primer dimer interference penalty score includes calculating the change in Gibbs free energy of the primer dimers formed by the forward primer and the reverse primer in the candidate primer pair or pairwise combinations of primers in different candidate primer pairs, and the primer dimer interference penalty score is calculated based on the degree of primer dimer extension determined by the change in Gibbs free energy of the primer dimer; Preferably, the number of the candidate primer pairs is represented by n, and P i and P j represent two of the candidate primer pairs, where i = 1, 2, 3, ..., n, j = 1, 2, 3, …, n, and i ≠ j; the Gibbs free energy change of the primer dimer formed by pairwise combination of the forward primer and the reverse primer in the candidate primer pair or the primers in different candidate primer pairs is ΔG, and the primer dimer interference penalty interaction i or the primer dimer interference penalty interaction when different candidate primer pairs coexist ij is obtained according to the following formula: Preferably, the primer dimer interference penalty score when different combinations of the candidate primer pairs coexist includes selecting the maximum primer dimer interference penalty score among the pairwise combinations of primers as the final penalty score.

3. The method according to any one of claims 1-2, characterized in that, Calculating the penalty score of each group of the candidate primer pairs or the penalty score when different combinations of the candidate primer pairs coexist further includes one or more of the number penalty score of non-specific amplicons and the nucleotide polymorphism site penalty score on the primer; Preferably, calculating the penalty score of each group of the candidate primer pairs further includes the GC content penalty score of the primer; Preferably, the number penalty score of non-specific amplicons includes calculating the number penalty score of non-specific amplicons in the background sequence; Preferably, the background sequence includes other non-target sequences in the test sample except the target sequence; Preferably, the GC content penalty score of the primer includes giving different penalty weights according to different content ranges of the GC content; More preferably, the GC content penalty score of the primer is calculated by the following formula: Among them, gc_score i is the GC content penalty of the primer; Preferably, the nucleotide polymorphism site penalty score on a single primer is calculated by the following formula: SNP i = 0.1 × the total number of SNPs on the primer + 1.2 × the number of SNPs in the 5 bases at the 3'-end of the primer Preferably, when there is a SNP at the last base at the 3' end of the primer, the primer pair is filtered out; Preferably, the nucleotide polymorphism site penalty score when the candidate primer pair or different combinations of the candidate primer pairs coexist is equal to the sum of the nucleotide polymorphism site penalty scores of all primers in the candidate or combination; Preferably, the penalty score of each group of the candidate primer pairs includes summing up the dimer interference penalty score, the number penalty score of non-specific amplicons, the GC content penalty score of the primer, and the nucleotide polymorphism site penalty score on the primer; and / or The penalty score when different combinations of the candidate primer pairs coexist includes summing up the total penalty score of the dimer interference penalty scores of pairwise combinations of primers in the different candidate primer pairs, the total penalty score of the number penalty scores of non-specific amplicons, and the total penalty score of the nucleotide polymorphism site penalty scores.

4. The method according to any one of claims 1 to 3, characterized in that, The penalty score matrix and primer coverage region matrix for calculating primer combinations include recording the penalties when each group of the candidate primer pairs or different combinations of the candidate primer pairs coexist through the costM matrix, and the primer coverage region matrix for calculating primers includes recording the regions covered by the primers through the positionM matrix; Preferably, the region covered by the positionM matrix recording primers includes recording the length of the region to be amplified as m, and initializing to generate a full-zero matrix positionM of m×n order; calculating the region S i covered by the candidate primer pair P k (k = 1, 2, 3,..., m), and generating a 0-1 matrix; Preferably, the penalty points recorded in the costM matrix when each group of the candidate primer pairs or different combinations of the candidate primer pairs coexist include initializing and generating an all-zero matrix costM of n×n order; traversing all candidate primer pair combinations Pi and P j (j < i), when i = j, replacing the element in the i-th row and j-th column of the matrix with costi; when i ≠ j, replacing the element in the i-th row and j-th column of the matrix with costi j ; Preferably, the calculation of the optimal primer combination using integer programming includes denoting all the primer pair selection combinations as x, calculating the sum of costM and x, and solving the integer programming through the constraint conditions. χ is the primer pair selection combination with the minimum sum value of costM after solving, and χ is the multiplex PCR primer combination; More preferably, the constraint condition is that each column of positionM×x is an integer not less than 1 and not greater than 2.

5. A device for designing multiplex PCR primers, characterized in that, Includes: A primer design module, configured to design or input a set of candidate primer pairs covering the entire target interval; A penalty score calculation module, including a penalty score unit configured to calculate the penalty score for each group of candidate primer pairs, a penalty score unit configured to calculate the penalty score when different combinations of candidate primer pairs coexist, a penalty score matrix unit configured to calculate the penalty score matrix of the primer combination, and a coverage region matrix unit configured to calculate the coverage region matrix of the primers; And An integer programming module, configured to calculate the optimal primer combination using integer programming; Among them, the calculation of the penalty score for each group of the candidate primer pairs or the penalty score when different combinations of the candidate primer pairs coexist in the primer pair penalty score unit is set to include calculating the primer dimer interference penalty score. The penalty score when different combinations of the candidate primer pairs coexist includes the penalty scores of pairwise combinations of primers in different candidate primer pairs.

6. The device according to claim 5, wherein Calculating the primer dimer interference penalty score includes calculating the change in Gibbs free energy of the primer dimer formed by the forward primer and the reverse primer in the candidate primer pair or pairwise combinations of primers in different candidate primer pairs. The primer dimer interference penalty score is calculated based on the degree of primer dimer extension determined by the change in Gibbs free energy of the primer dimer; Preferably, the number of the candidate primer pairs is represented by n, and P i and P j represent two of the candidate primer pairs, where i = 1, 2, 3, ..., n, j = 1, 2, 3, …, n, and i ≠ j; the Gibbs free energy change of the primer dimer formed by pairwise combination of the forward primer and the reverse primer in the candidate primer pair or primers in different candidate primer pairs is ΔG, and the primer dimer interference penalty interaction i or the primer dimer interference penalty interaction when different candidate primer pairs coexist ij is obtained according to the following formula: Preferably, the primer dimer interference penalty score when different combinations of the candidate primer pairs coexist includes selecting the maximum primer dimer interference penalty score among the pairwise combinations of primers as the final penalty score.

7. The device according to any one of claims 5-6, characterized in that The calculation of the penalty score for each group of the candidate primer pairs or the penalty score when different combinations of the candidate primer pairs coexist in the primer pair penalty score unit is also set to include one or more of the penalty score for the number of non-specific amplicons and the penalty score for nucleotide polymorphic sites on the primers; Preferably, the calculation of the penalty score for each group of the candidate primer pairs further includes the penalty score for the GC content of the primers; Preferably, the penalty score for the number of non-specific amplicons includes calculating the penalty score for the number of non-specific amplicons in the background sequence; Preferably, the background sequence includes other non-target sequences in the test sample except the target sequence; Preferably, the penalty score for the GC content of the primers includes giving different penalty score weights according to different content ranges of the GC content; More preferably, the penalty score for the GC content of the primers is calculated through the following formula: Among them, gc_score i is the GC content penalty of the primer; Preferably, the penalty score for nucleotide polymorphic sites on a single primer is calculated through the following formula: SNP i = 0.1 × the total number of SNPs on the primer + 1.2 × the number of SNPs in the 5 bases at the 3' end of the primer Preferably, when there is a SNP at the last base at the 3'-end of the primer, this primer pair is filtered out; Preferably, the nucleotide polymorphism site penalty when the candidate primer pairs or different combinations of the candidate primer pairs coexist is equal to the sum of the nucleotide polymorphism site penalties of all the primers within the candidate or combination; Preferably, the penalty score for each group of the candidate primer pairs is obtained by adding up the dimer interference penalty score, the non-specific amplicon number penalty score, the primer GC content penalty score, and the nucleotide polymorphism site penalty score on the primer; and / or The penalty score when different combinations of the candidate primer pairs coexist is obtained by adding up the total dimer interference penalty score of pairwise combinations of the primers in different candidate primer pairs, the total non-specific amplicon number penalty score, and the total nucleotide polymorphism site penalty score.

8. The device according to any one of claims 5 to 7, characterized in that The penalty score matrix for calculating the primer combination in the penalty score matrix unit is set to include recording the penalty scores when each group of the candidate primer pairs or different combinations of the candidate primer pairs coexist through the costM matrix, and the coverage area matrix for calculating the primer in the coverage area matrix unit is set to include recording the area covered by the primer through the positionM matrix; Preferably, the region covered by the primer recorded in the positionM matrix includes the length of the region to be amplified, denoted as m, and a zero matrix positionM of m×n order is initialized and generated; calculate the region S covered by the candidate primer pair P i covered by k (k = 1, 2, 3,..., m), and generate a 0-1 matrix; Preferably, the penalty points recorded in the costM matrix when each group of the candidate primer pairs or different combinations of the candidate primer pairs coexist include initializing and generating an all-zero matrix costM of order n×n; traversing all combinations of candidate primer pairs P i and P j (j < i), when i = j, replacing the element in the i-th row and j-th column of the matrix with cost i ; when i ≠ j, replacing the element in the i-th row and j-th column of the matrix with cost ij ; Preferably, the integer programming module includes recording all primer pair selection combinations as x, calculating the sum of costM and x, and performing integer programming solution through constraint conditions. χ is the primer pair selection combination with the minimum sum of costM after solution, and χ is the multiplex PCR primer combination; More preferably, the constraint condition is that each column of positionM×x is an integer not less than 1 and not greater than 2.

9. A computer-readable storage medium, characterized in that, The storage medium includes a stored program, wherein when the program runs, it controls the device where the storage medium is located to execute the multiplex PCR primer design method according to any one of claims 1-4.

10. A processor, characterized in that, The processor is used to run the program, wherein when the program runs, it executes the multiplex PCR primer design method according to any one of claims 1-4.

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