A method for designing and evaluating multiplex digital PCR primer probes for target pathogens or drug resistance genes

Abstract

The present application relates to the field of bioinformatics technology, and specifically discloses a method for designing and evaluating multiple digital PCR primer probes for target pathogens or drug-resistant genes. The present application first scores and sorts all primer probes for a single target pathogen or drug-resistant gene through a custom algorithm, and then adds the better primer probes for a single target pathogen or drug-resistant gene in a manner of increasing the number of targets or multiplicity, and evaluates the secondary structure and primer amplification specificity of the entire combination system after each addition of primer probes, until a primer probe combination that meets the requirements is screened. The present application can realize the automatic combination design of primer probes for high-weight target pathogens or drug-resistant genes.

Description

Technical Field

[0001] The present application relates to the technical field of bioinformatics, and in particular to a multiplex digital PCR primer probe design and evaluation method, model and application thereof for target pathogens or drug-resistant genes. Technical Background

[0002] Compared with traditional culture and other detection methods, the detection and identification of infectious pathogens based on NGS sequencing technology are currently mainly used in clinical practice: mNGS (metagenomic next generation sequencing) and tNGS (multiple amplification + NGS). mNGS detection has the advantages of high sensitivity, specificity and wide coverage of pathogens. tNGS is based on the amplification of target pathogen-specific sequences, and the amount of subsequent sequencing data required is less than that of mNGS, so the cost is lower and it has a higher cost-effectiveness. However, whether it is mNGS or tNGS, due to the limitation of sequencing time, the detection time of both cannot achieve D0 day report. Therefore, multiple digital PCR (ddPCR) with faster timeliness and controllable cost may become a more preferred pathogen detection method in the future.

[0003] At present, some pathogen or drug resistance gene detection products based on multiplex amplification technology have been seen on the market, and different products can detect different contents and targets. Thermo Fisher TAC multiplex qPCR is for plasma specimens and can detect 48 targets; Filmarray 2.0BCID2 Panel is for blood culture and can detect 43 targets; Linghang dPCR, which currently occupies the main market in China, is for plasma specimens and can detect 24 targets (the results can be obtained in about 4 hours).

[0004] The design requirements for multiplex PCR are different depending on the number of multiplexes. Unlike traditional 4-5 multiplex PCR, higher multiplex or super multiplex PCR technology has higher design requirements and is more difficult. Multiplex digital PCR technology, in addition to considering the design of single primers and primer combination effects, also requires the design of probe sequences. Summary of the invention

[0005] In order to solve the above technical problems, the present application has developed a primer probe design and evaluation method for target pathogens or drug-resistant genes, which is suitable for ultra-multiplex digital PCR. Through this method, the present application designs a primer probe sequence combination of 17 layers for 11 drug-resistant genes and 11 layers for 11 pathogenic bacteria, and the experimental verification shows that the effect is obviously superior.

[0006] Therefore, this application includes at least the following objectives:

[0007] The first purpose of this application is to seek a method for designing and evaluating primer probes for super-multiplex digital PCR targeting target pathogens or drug resistance genes;

[0008] The second objective of the present application is to seek a model for the design and evaluation of primer probes for super-multiplex digital PCR targeting target pathogens or drug-resistant genes.

[0009] In order to achieve the above objectives, this application specifically proposes the following specific technical solutions.

[0010] The present application first provides a method for designing and evaluating primer probes for super-multiplex digital PCR of target pathogens or drug resistance genes, comprising the following steps:

[0011] 1) Design and select primer and probe sequences for a single target pathogen or a single drug resistance gene;

[0012] 2) Screen and select primer-probe sequence combinations for all target pathogens or drug-resistant genes.

[0013] In some aspects, the designing and selecting in step 1) comprises the following steps:

[0014] a) Based on all specific core genome sequences of a single target pathogen or a single drug resistance gene template sequence, primers were designed using Primer3 to obtain batch primer pairs and amplicon sequences;

[0015] b) Use MFEprimer to evaluate the batch primer sequences and evaluate and screen suitable probe sequences in other non-primer regions on the amplicon for each primer pair;

[0016] c) Evaluate primer amplification specificity based on human reference genome sequences, clinical environment-related microbial reference genome sequences, and the CARD drug resistance gene database;

[0017] d) Use a custom formula to calculate the score of the primers / probes and sort the primers / probes from low to high according to the score results.

[0018] The pathogen-specific core genome sequence described in this article refers to a sequence fragment that is carried by the genome of each strain in the target pathogen and is unique to the target pathogen relative to other bacteria; the drug-resistant gene template sequence refers to a single drug-resistant gene that may have different variants due to its sequence polymorphism (for example, the KPC gene has multiple subtypes such as KPC-2, KPC-3, and KPC-4). When designing primers, the subtype with a higher frequency of occurrence of the drug-resistant gene is selected as the representative sequence of the gene, that is, the template sequence.

[0019] Further, the step b) is specifically as follows: using MFEprimer to evaluate the batch primer sequences, filtering out the primers whose product tm obtained by MFEprimer evaluation has a large difference (>19°C) with the primer tm; then filtering out the primer pairs with a high variation frequency (>1%) and a variation site number >2 or a single variation site at the 3' end on the primer sequence based on the nucleotide variation information on the template sequence; for the remaining primer pairs and their amplicons, intercepting a certain length (18 to 30 bp) of sequence from different positions close to the F primer or R primer end, and evaluating whether it can be used as a probe; if a qualified probe sequence is found on the amplicon, retain the current primer, otherwise filter out the corresponding primer;

[0020] Preferably, the probe sequence evaluation parameters include:

[0021] a. Sequence base composition: the first base at the 5' end is not G, the sequence does not contain GGGG and AAAAAA, and the GC content is in the range of 30%-80%;

[0022] b. The tm value of the sequence is in the range of 64-72°C, and the difference with the tm of the primer is in the range of 5-10°C;

[0023] c. MFEprimer evaluation requires that the sequence itself has no hairpin structure and dimer structure, and does not form dimers with primers;

[0024] d. The number of sites with a high frequency of variation (>1%) in the sequence does not exceed 2.

[0025] Furthermore, the evaluation in step c) is:

[0026] For the target pathogen, MFEprimer is used to evaluate the amplification specificity of each primer relative to the human reference genome and clinically relevant microorganisms, and a score is given based on the results;

[0027] Preferably, the scoring rule is: if a human sequence is amplified, it is scored as 1 point, and if the amplicon length is short (<=300bp), it is scored as 2 points; if non-specific other pathogens are amplified, it is scored as 0.25 points, and if the amplicon length is short (<=300bp), it is scored as 0.5 points; if there is no non-specific amplification, it is scored as 0 points;

[0028] For drug-resistant genes, in addition to evaluating the amplification specificity relative to the human reference genome and microbial species, it is also necessary to evaluate the specificity of the target drug-resistant gene relative to other genes; the other genes are other drug-resistant genes in the CARD drug-resistant gene database except the target drug-resistant gene; similarly, if there is nonspecific amplification, it is scored as 0.25 points, and if the amplicon is short (<=300bp), it is scored as 0.5 points.

[0029] Furthermore, the step d) is specifically as follows: using a custom formula to calculate the scores of the primers / probes, and sorting them according to the score results;

[0030]

[0031] The scoring formula is:

[0032] Among them, Penalty Score is the comprehensive score of primer probe, F is various factors, and Score is the score of the corresponding factor; the comprehensive score of each primer / probe is obtained by calculation, and the primers / probes are sorted from low to high according to the comprehensive score.

[0033] Preferably, the scoring rules of the various factors are as follows:

[0034]

[0035]

[0036] In some aspects, step 2) comprises the following steps:

[0037] i) For all target pathogens or drug-resistant genes, sort them from high to low in importance or from low to high in the number of candidate available primer pairs, and evaluate and gradually expand the combination of all primer probes to meet the requirements of no secondary structure and non-specific amplification by adding one target primer probe at a time, and screen the primer probe combinations of all targets;

[0038] ii) Evaluate the effect of each primer / probe pair for the primer-probe combination obtained, requiring that the primers and probes have no secondary structure and that the scores of the primers and the products are not less than 600;

[0039] iii) Design degenerate bases on primers or probes based on template variation information; evaluate the genome coverage of each primer pair for pathogens.

[0040] Furthermore, the step i) is specifically as follows:

[0041] Starting with the primer probe with the best score for the first target pathogen or drug-resistant gene, add the primer probe with the best score for the second target to the combination, and use MFEprimer software to evaluate the secondary structure and primer amplification specificity of the 2X primer probe combination; if there is no secondary structure and nonspecific amplification, add the best primer probe for the third target, otherwise pick other candidate primer probes for the second target and add them to the combination for further evaluation; in this way, gradually complete the selection and evaluation of primer probe combinations for all targets; if a suitable primer probe cannot be found for a certain target during the process, promote this target to the first place, and repeat the operation until the primer probe combinations that meet the requirements for all targets are automatically screened out.

[0042] Furthermore, the step ii) is specifically as follows:

[0043] ii) For the primer-probe combination obtained by screening, use Oligo7 software to evaluate the effect of each primer / probe pair, requiring that the primers and probes have no secondary structure and the scores of the products are not less than 600.

[0044] Furthermore, the step iii) is specifically as follows:

[0045] iii) Design degenerate bases on primers or probes based on template variation information; use the MFEprimer online tool to evaluate the genome coverage of each primer pair for pathogens.

[0046] In some aspects, the above design and evaluation method further comprises step 3):

[0047] Targeting pathogens or drug-resistant genes, pure bacterial strains or synthetic plasmid sequences are used to conduct qPCR experiments and multiplex digital PCR experiments to verify the effectiveness of primers and probes.

[0048] The present application also provides a model for designing and evaluating primer probes for super-multiplex digital PCR of target pathogens or drug resistance genes, the model comprising the following modules:

[0049] Module 1) is used to design and select primer probe sequences for a single target pathogen or drug resistance gene based on the pathogen-specific core genome sequence or drug resistance gene template sequence;

[0050] Module 2) is used to screen and evaluate the selection of primer-probe sequence combinations for all target pathogens or drug-resistant genes.

[0051] In some aspects, the designing and selecting in module 1) includes:

[0052] a) Based on all specific core genome sequences or drug resistance gene template sequences of a single target pathogen, primers were designed using Primer3 to obtain batch primer pairs and amplicon sequences;

[0053] b) Use MFEprimer to evaluate the batch primer sequences and evaluate and screen suitable probe sequences in other non-primer regions on the amplicon for each primer pair's amplicon sequence;

[0054] c) Evaluate primer amplification specificity based on human reference genome sequences, clinical environment-related microbial reference genome sequences, and the CARD drug resistance gene database;

[0055] d) Use a custom formula to calculate the scores of primers / probes and sort them according to the scoring results, and sort the primers / probes from low to high according to the comprehensive scores.

[0056] In some aspects, the screening and evaluation in module 2) includes the following aspects:

[0057] i) For all target pathogens or drug resistance genes, sort them from high to low in terms of clinical importance or from low to high in terms of the number of candidate available primer pairs, and evaluate and gradually expand the combinations of all primer probes to meet the requirements of no secondary structure and non-specific amplification by adding one target primer probe at a time, and screen the primer probe combinations for all targets;

[0058] ii) Evaluate the effect of each primer / probe pair for the primer-probe combination obtained, requiring that the primers and probes have no secondary structure and that the scores of the primers and the products are not less than 600;

[0059] iii) Design degenerate bases on primers or probes based on template variation information; evaluate the genome coverage of each primer pair for pathogens.

[0060] The present application also provides an electronic device, comprising: a processor and a memory; the processor and the memory are connected, wherein the memory is used to store a computer program, and the processor is used to call the computer program to execute any of the methods described above.

[0061] The present application also provides a computer storage medium, wherein the computer storage medium stores a computer program, wherein the computer program includes program instructions, and when the program instructions are executed by a processor, the method described in any of the above

[0062] Beneficial technical effects of this application:

[0063] 1) There are few design methods in the prior art that can design probes for a large number of primers in batches (especially for target pathogens or drug-resistant genes). This application combines public software for primer design and evaluation, and independently develops an automated method based on appropriate parameter settings that can design probes for target pathogens or drug-resistant genes in batches. At the same time, it can complete the secondary structure and amplification specificity evaluation and selection filtering of primer probe sequences.

[0064] 2) This application first scores and ranks all primer probes of a single target pathogen or drug resistance gene through a custom algorithm, then adds the better primer probes of a single target pathogen or drug resistance gene in a way of increasing the number of targets or multiplicity, and evaluates the secondary structure and primer amplification specificity of the entire combination system after each addition of primer probes, until a primer probe combination that meets the requirements is screened. This method can realize the automatic combination design of primer probes with a higher multiplicity. BRIEF DESCRIPTION OF THE DRAWINGS

[0065] Figure 1 , technical roadmap of this application.

[0066] Figure 2 , VIM gene clustering results. DETAILED DESCRIPTION

[0067] The embodiments of the present application will be described in detail below in conjunction with the examples, but it will be appreciated by those skilled in the art that the following examples are only used to illustrate the present application and should not be considered as limiting the scope of the present application. In the examples, if no specific conditions are specified, the conditions are carried out according to normal conditions or manufacturer recommendations. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be purchased from the market.

[0068] Definition of some terms

[0069] Unless otherwise defined below, the meaning of all technical terms and scientific terms used in the specific embodiments of the present application is intended to be the same as those generally understood by those skilled in the art. Although it is believed that the following terms are well understood by those skilled in the art, the following definitions are still set forth to better explain the present application.

[0070] As used in this application, an indefinite or definite article used when referring to a singular noun eg "a" or "an", "the" includes a plural of that noun.

[0071] As used in this application, the terms "comprises", "comprising", "having", "containing" or "involving" are inclusive or open-ended and do not exclude other unrecited elements or method steps. The term "consisting of" is considered a preferred embodiment of the term "comprising". If a group is defined below as comprising at least a certain number of embodiments, this should also be understood to disclose a group that preferably consists of only these embodiments.

[0072] The term "approximately" in this application indicates an accuracy range that can be understood by those skilled in the art and still ensures the technical effect of the characteristic in question. The term generally indicates ±10%, preferably ±5%, of the indicated value.

[0073] In addition, the terms first, second, third, (a), (b), (c), and the like in the specification and claims are used to distinguish similar elements and are not necessarily required to describe a sequential or chronological order. It should be understood that the terms so used are interchangeable under appropriate circumstances, and the embodiments described in this application can be implemented in other sequences than those described or illustrated in this application.

[0074] The above terms or definitions are provided only to help understand the present application. These definitions should not be construed as having a scope less than that understood by those skilled in the art.

[0075] The present application is described below in conjunction with specific embodiments.

[0076] Example 1: Design and digital PCR verification of 17-plex primer-probe combinations for 11 drug resistance genes

[0077] This embodiment is based on the method steps of the aforementioned application (see the basic roadmap for details) Figure 1 ) High-density digital PCR primer probes were designed for 11 drug-resistant genes, as follows.

[0078] 1. For the 11 common clinical drug-resistant genes, select appropriate reference sequences (as shown in Table 1-1 below), and obtain variation information on the reference sequences through strain genome comparison detection (as shown in Table 1-2).

[0079] 1.1 Detection and frequency statistics of target drug-resistant genes based on strain genomes downloaded from public libraries. All genome sequences of eight common clinical bacteria (Klebsiella pneumoniae, Escherichia coli, Enterobacter cloacae complex, Acinetobacter baumannii, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus pneumoniae, and Enterococcus faecium) were downloaded from the PARIC database (https: / / www.bv-brc.org), and the CARD library (https: / / card.mcmaster.ca) was downloaded as a reference library for drug-resistant genes. The strain genome sequences were aligned using blastn software (blastn-task blastn-evalue 1e-5-num_threads 6-num_alignments 10000-outfmt 6, for each alignment region of the genome query, the best alignment was selected as the final alignment result) to detect the detection frequencies of 11 target drug-resistant genes, and the query sequences of the corresponding aligned regions were intercepted.

[0080] 1.2 Select the reference sequence of each gene based on the clustering results of sequence similarity between its subtypes or variants. All subtypes or variant sequences of the 11 drug-resistant genes were downloaded from the CARD public drug resistance database, and then all subtypes or variant sequences within each gene were compared to obtain homology values, and clustered based on sequence similarity (the default threshold is 90%). According to the clustering results, the corresponding subtype or variant with higher frequency was selected from each cluster as the reference sequence (such as Figure 2 ).

[0081] 1.3 The query sequence obtained in step 1.1 is compared with the selected reference sequence to detect the variation information on the reference sequence. Use blastn to extract the query sequence (blastn-taskblastn-evalue 1e-5-num_threads 6-outfmt 0, the default homology threshold is 90%), and then parse the m0 format file into m6 format, and obtain the variation information on each reference sequence, and select the best alignment as the final alignment result for each alignment region of the genomic query. Finally, the coverage depth of the reference sequence and the SNP and InDel variation information can be statistically obtained.

[0082] Table 1-1 11 drug-resistant genes and their template typing.

[0083]

[0084]

[0085] Table 1-2 Drug resistance gene reference sequence variation information

[0086] Target genes Base position Depth of coverage Reference base SNP insert Missing Total frequency mea 1 2383 A 0 0 0 2385 mea 2 2383 T 0 0 0 2385 mea 3 2383 G 0 0 0 2385 mea 4 2383 A 0 0 0 2385 ... mea 675 2382 T T->A:527 0 0 2385 mea 676 2382 G 0 0 0 2385 mea 677 2382 A 0 0 0 2385 mea 678 2382 T 0 0 0 2385 mea 679 2382 T 0 0 0 2385 mea 680 2382 T 0 0 0 2385 mea 681 2382 C 0 0 0 2385 mea 682 2382 G G->A:40 0 0 2385 ... mea 713 2382 C C->T:6 0 0 2385 mea 714 2382 A 0 0 0 2385 mea 715 2382 A A->G:2320 0 0 2385 mea 716 2382 A 0 0 0 2385 ... mea 736 2382 G 0 0 0 2385 mea 737 2382 A A->G:1355 0 0 2385 mea 738 2382 A 0 0 0 2385 ...

[0087] 2. Design primers and probes based on the reference sequences of each drug-resistant gene template, and evaluate their secondary structures and primer specificity. For each drug-resistant gene, primers were designed using primer3 software based on the selected template reference sequence, and probe sequences were designed using a self-written program. MFEprimer software was used to evaluate the secondary structure and tm value of primer probes, and the initial selection of primer probes was completed. Then, MFEprimer was used to evaluate the specificity of primers based on the human reference genome, the constructed microbial reference genome, and the CARD drug resistance database.

[0088] 2.1 Based on the drug-resistant gene template sequence, use the python language primer3 package to design enough primer sequences. Parameter settings: --primer_num 20000--min-amplicon-length 80--max-amplicon-length150--optimal-amplicon-length 100--min-primer-length 18--max-primer-length24--optimal-primer-length 20--min-primer-melting-temp 58--max-primer-melting-temp62--optimal-primer-melting-temp 60--min-primer-gc 40--max-primer-gc 60--optimal-primer-gc 50--max-primer-poly-n 5--monovalent-concentration 50--divalent-concentration 1.5--dntp-concentration 0.25--primer-concentration 50.

[0089] 2.2 Based on the amplicon sequence of each primer pair, probes are designed in the non-primer region on the amplicon, and MFEprimer is used to evaluate and select qualified primer probe sequences. First, the primers that do not meet the requirements are filtered out, that is, the requirements are: the length of the amplified product is within 80-120bp, the difference between the product tm and the primer tm is <=19°C, the difference between the length of the F and R primers is <=4bp, the difference between the tm of the F and R primers is <=2°C, the base sites with an incidence rate of >=0.01 on the primers are recorded as variant sites, the maximum number of variant sites allowed on the primers is <=2, and the first base at the 5' end of the primer and the 5 base sites at the 3' end cannot have variants. Then, for the primer pairs and amplicons that meet the requirements, sequences with a length of between 18 and 30bp are intercepted from different positions close to the F primer or R primer end to evaluate whether they can be used as probes. The probe sequence evaluation parameters include:

[0090] a. Sequence base composition: The first base at the 5' end must not be G, the sequence must not contain GGGG and AAAAAA, and the GC content must be within the range of 30%-80%;

[0091] b. The tm value of the sequence is required to be within the range of 64-72°C, and the difference with the tm of the primer is within the range of 5-10°C;

[0092] c. MFEprimer evaluation (mfeprimer-3.3.0-linux-amd64 dimer-d-3-m 1) requires that the sequence itself has no hairpin structure and dimer structure, and does not form dimers with the primer.

[0093] d. The number of mutation sites with a high frequency (default > 1%) in the sequence is required to be no more than 2, especially within the 5 bases close to the 3' end.

[0094] 2.3 Based on the human reference genome sequence (GRCh8), the clinical environment-related microbial reference genome sequence, and the CARD drug resistance gene database, the specificity of primer amplification was evaluated. MFEprimer (mfeprimer-3.3.0-linux-amd64spec) was used to evaluate the amplification specificity of each primer relative to the human reference genome and clinically relevant microorganisms, and scores (penalties) were given based on the results. Scoring rules: if human sequences were amplified, it was scored as 1, and if the amplicon length was short (default <=300bp), it was scored as 2; if nonspecific other pathogens were amplified, it was scored as 0.25, and if the amplicon length was short (default <=300bp), it was scored as 0.5; if there was no nonspecific amplification, it was scored as 0. In addition to evaluating the amplification specificity relative to the human reference genome and microbial species, it is also necessary to evaluate the specificity of the target drug resistance gene relative to other genes. Similarly, if there is nonspecific amplification, it is scored as 0.25, and if the amplicon is short (default <=300bp), it is scored as 0.5.

[0095] 2.4 Use a custom formula to calculate the score of primers / probes and sort them according to the score results. The scoring formula is:

[0096] Among them, Penalty Score is the comprehensive score of the primer probe, F is the relevant factors, and Score is the score of the factors. The factors and their scoring rules are shown in Table 1-3:

[0097] Table 1-3 Primer probe sequence scoring rules

[0098]

[0099] In summary, the comprehensive score (i.e., penalty value) of each primer / probe is calculated, and then the primers / probes are sorted from low to high according to the comprehensive score.

[0100] 3. For 11 drug-resistant genes, MFEprimer was used to evaluate and select primer-probe sequence combinations by gradually increasing primers and probes, and Oligo7 software was used for secondary evaluation.

[0101] 3.1 For all 11 drug-resistant genes, they were sorted from low to high according to the number of candidate available primer pairs, and then one target primer probe was added each time to evaluate and gradually expand the combination of all primer probes to meet the requirements of no secondary structure and nonspecific amplification. Specifically, first start with the primer probe with the best score for the first drug-resistant gene (i.e., the smallest score in step 2.4), add the primer probe with the best score for the second drug-resistant gene to the combination, and use MFEprimer software to evaluate the secondary structure of the 2X primer probe combination (evaluation of hairpin structure: mfeprimer-3.3.0-linux-amd64 hairpin-d-3; evaluation of primer dimer: mfeprimer-3.3.0-linux-amd64 dimer-d-3) and primer amplification specificity. If there is no secondary structure and nonspecific amplification, then add the best primer probe for the third drug-resistant gene, otherwise pick other candidate primer probes for the second drug-resistant gene and add them to the combination for further evaluation. In this way, the selection and evaluation of primer-probe combinations for all targets are completed step by step. If a certain drug-resistant gene cannot find a suitable primer-probe during the step-by-step evaluation process, this drug-resistant gene will be promoted to the first place, and the above operation will be repeated until the primer-probe combinations of all 11 drug-resistant genes that meet the requirements are automatically screened out.

[0102] 3.2 For the primer-probe combination obtained above, the Oligo7 software was finally used to evaluate the effect of each primer / probe pair, requiring that the primers and probes had no secondary structure and the scores with the product were not less than 600, otherwise the primers or probes were replaced according to 3.1. Finally, the sequences and scoring tables of 17 primer-probe combinations for 11 drug-resistant genes were obtained as shown in Tables 1-4 and 1-5 below.

[0103] Table 1-4 17-plex primer-probe sequence combinations for 11 drug resistance genes

[0104]

[0105]

[0106]

[0107] Table 1-5 Evaluation results of Oligo7 with 17 primers and probe sequences for 11 drug resistance genes

[0108]

[0109] 4. Use bacterial strains or plasmids to synthesize drug-resistant gene sequences to conduct qPCR experiments and digital PCR experiments to verify the effectiveness of primer probes.

[0110] 4.1 Collect strains or synthetic plasmids carrying target drug-resistant genes. Collect and culture isolate strains from clinical sites for WGS sequencing. By analyzing the presence of drug-resistant genes in the genome, find strains carrying target drug-resistant genes as experimental materials. For some low-frequency drug-resistant genes, if it is difficult to find clinical culture isolates carrying target drug-resistant genes, obtain the target drug-resistant gene sequence by synthetic plasmids (such as sending them to Sangon Biotech (Shanghai) Co., Ltd. for plasmid synthesis).

[0111] 4.2 For the 17-plex primer probes, the fluorescence quantitative PCR platform and the digital PCR platform were used to conduct experimental verification of the effect. For each target gene, the primer effect was first tested using the Hangzhou Biori Gene 9660 fluorescence quantitative PCR platform (melting curve method), and a single primer pair and a 17-plex primer combination were used for testing. The single primer and the 17-plex primer combination were required to be compared, and the δCt value difference was within ±2 and the δ target peak was within ±250 to be qualified. Then, the single primer pair test was first performed using the Pilot fully automatic droplet digital PCR system (AD3207) (probe method; limited to a maximum of 7 plexes), and the concentration of drug-resistant gene nucleic acid could be quantitatively obtained. For plasmids, the difference between the known concentration and the digital PCR determination concentration can be compared, and the δ concentration difference within ±50% is considered qualified. Finally, due to the limitation that the Pilot digital PCR system has a maximum of 7 plexes on a chip, the 17 primer probes were divided into 3 groups according to 6 primer probes as a combination for testing the 6-plex primer probe combination, and the detection sensitivity (LOD) of each target gene was determined. According to the quantitatively measured drug-resistant gene nucleic acid concentration, each drug-resistant gene nucleic acid was diluted to 6e+3 copies / ml, and then the 6 drug-resistant gene nucleic acids in the combination were mixed and diluted 2 times to obtain 5 gradient concentrations (1000 / 500 / 250 / 125 / 62.5 copies / ml) of nucleic acid samples. Then, a 6-plex primer probe test was performed based on the 5 gradient concentrations of nucleic acid samples to evaluate the detection effect and detection sensitivity. All experimental results are shown in Tables 1-6.

[0112] Table 1-6 Test results of 17-plex primer-probe combination experiment

[0113]

[0114]

[0115]

[0116] Conclusion: For 17 drug-resistant gene targets, single primers and 17-plex primer combinations were first tested using fluorescent quantitative PCR. The results showed that for each primer, the CT value and target peak value of single amplification and combined primer amplification were consistent, indicating that the 17-plex primer combination amplification effect was good; then, the single primer probe sequence was further tested using the digital PCR platform. The results showed that all 17 primer probe sequences could work normally and sequence the concentration values ​​of target genes, and the probe quantitative values ​​of 7 genes such as VIM-2 and OXA-48 using plasmids as test materials were consistent with the plasmid standard concentration values; finally, the 17-plex primer probe combination was used, and the target gene detection sensitivity (LOD) of each primer probe was determined using the digital PCR platform. The results showed that the measured sensitivity was mainly 62.5 or 125 copies / ml.

[0117] In summary, the 17-plex primer-probe combination designed for target KPC, NDM and other drug-resistant genes can work normally, and the minimum detection limit is 62.5 or 125 copies / ml.

[0118] Example 2: Design and digital PCR verification of 11-plex primer-probe combinations for 11 bacteria

[0119] This example designs high-density digital PCR primers and probes for 11 bacteria based on the method steps of the present application, as follows.

[0120] 1. For 11 common clinical bacteria (as shown in Table 2-1), based on the specific core genome sequences and their variation information that have been screened, use primer3 software to design enough primer sequences, and then combine MFEprimer software to design and evaluate probe sequences, and select primer probe sequences with no secondary structure and less than 2 variation sites.

[0121] Table 2-1 List of 11 pathogenic bacteria

[0122]

[0123]

[0124] 1.1 Based on the target pathogen-specific core genome template sequence, use the python language primer3 package to design enough primer sequences. Parameter settings: --primer_num 20000--min-amplicon-length 80--max-amplicon-length 150--optimal-amplicon-length 100--min-primer-length 18--max-primer-length 24--optimal-primer-length 20--min-primer-melting-temp 58--max-primer-melting-temp 62--optimal-primer-melting-temp 60--min-primer-gc 40--max-primer-gc 60--optimal-primer-gc 50--max-primer-poly-n 5--monovalent-concentration 50--divalent-concentration 1.5--dntp-concentration 0.25--primer-concentration 50.

[0125] 1.2 Based on the amplicon sequence of each primer pair, probes are designed in the non-primer region on the amplicon, and MFEprimer is used to evaluate and select qualified primer probe sequences. First, the primers that do not meet the requirements are filtered out, that is, the requirements are: the length of the amplified product is within 80-120bp, the difference between the product tm and the primer tm is <=19°C, the difference between the length of the F and R primers is <=4bp, the difference between the tm of the F and R primers is <=2°C, the base sites with an incidence rate of >=0.01 on the primers are recorded as variant sites, the maximum number of variant sites allowed on the primers is <=2, and the first base at the 5' end of the primer and the 5 base sites at the 3' end cannot have variants. Then, for the primer pairs and amplicons that meet the requirements, sequences with a length of between 18 and 30bp are intercepted from different positions near the F primer or R primer end to evaluate whether they can be used as probes. The probe sequence evaluation parameters include:

[0126] a. Sequence base composition: the first base at the 5' end must not be G, the sequence must not contain GGGG and AAAAAA, and the GC content must be within the range of 30%-80%;

[0127] b. The tm value of the sequence is required to be within the range of 64-72°C, and the difference with the tm of the primer is within the range of 5-10°C;

[0128] c. MFEprimer evaluation (mfeprimer-3.3.0-linux-amd64 dimer-d-3-m 1) requires that the sequence itself has no hairpin structure and dimer structure, and does not form dimers with the primer.

[0129] d. The number of mutation sites with a high frequency (default > 1%) in the sequence is required to be no more than 2, especially within the 5 bases close to the 3' end.

[0130] 1.3 Based on the human reference genome sequence (GRCh8) and the clinical environment-related microbial reference genome sequence, the specificity of primer amplification was evaluated. MFEprimer (mfeprimer-3.3.0-linux-amd64 spec) was used to evaluate the amplification specificity of each primer relative to the human reference genome and clinically relevant microorganisms, and scores (penalties) were given based on the results. Scoring rules: Amplification of human sequences was scored as 1, and amplicon length was short (default <=300bp) was scored as 2; amplification of nonspecific other pathogens was scored as 0.25, and amplicon length was short (default <=300bp) was scored as 0.5; no nonspecific amplification was scored as 0.

[0131] 1.4 Use a custom formula to calculate the score of primers / probes and sort them according to the score results. The scoring formula is:

[0132] Penalty Score is the comprehensive score of the primer probe, F is the relevant factors, and Score is the score of the factors. The factors and their scoring rules are shown in Table 2-2.

[0133] Table 2-2 Primer probe sequence scoring rules

[0134]

[0135]

[0136] In summary, the comprehensive score (i.e., penalty value) of each primer / probe is calculated, and then the primers / probes are sorted from low to high according to the comprehensive score.

[0137] 2. For 11 pathogenic bacteria, MFEprimer was used to evaluate and select primer-probe sequence combinations by gradually adding primers and probes, and Oligo7 software was used for secondary evaluation.

[0138] 2.1 For all 11 pathogens, sort them from low to high according to the number of candidate available primer pairs, and then evaluate and gradually expand the combination of all primer probes to meet the requirements of no secondary structure and nonspecific amplification by adding one target primer probe at a time. Specifically, start with the primer probe with the best score for the first pathogen (i.e., the smallest score in step 2.4), add the primer probe with the best score for the second pathogen to the combination, and use MFEprimer software to evaluate the secondary structure of the 2X primer probe combination (evaluation of hairpin structure: mfeprimer-3.3.0-linux-amd64 hairpin-d-3; evaluation of primer dimer: mfeprimer-3.3.0-linux-amd64 dimer-d-3) and primer amplification specificity. If there is no secondary structure and nonspecific amplification, add the best primer probe of the third pathogen, otherwise pick other candidate primer probes of the second pathogen and add them to the combination for evaluation. In this way, the selection and evaluation of primer probe combinations for all targets are gradually completed. If a certain drug-resistant gene cannot find a suitable primer probe during the step-by-step evaluation process, this drug-resistant gene will be promoted to the first place, and the above operation will be repeated until the primer probe combination of all 11 pathogens that meet the requirements is automatically screened out.

[0139] 2.2 For the primer-probe combination obtained above, the Oligo7 software was finally used to evaluate the effect of each primer / probe pair, requiring that the primers and probes had no secondary structure and the scores with the product were not less than 600, otherwise the primers or probes were replaced according to 2.1. Finally, the sequences and scores of the 11-plex primer-probe combination for 11 pathogens were obtained as shown in Tables 2-3 and 2-4.

[0140] Table 2-3 11-plex primer-probe sequence combinations for 11 pathogenic bacteria

[0141]

[0142]

[0143]

[0144] Table 2-4 Evaluation results of 11 primer probe sequences Oligo7 for 11 pathogenic bacteria

[0145]

[0146]

[0147] Note: Genome coverage can be assessed using an online tool (https: / / m4.igenetech.com / cov).

[0148] 3. Use pure bacterial strains to conduct qPCR experiments and digital PCR experiments to verify the effectiveness of primer probes.

[0149] 3.1 The target bacteria strains isolated from clinical samples were collected and cultured as sample materials. The concentrations of Acinetobacter baumannii, Klebsiella pneumoniae, and Pseudomonas aeruginosa strains were known, but the concentrations of others were not measured.

[0150] 3.2 For the 11-plex primer probe, the fluorescence quantitative PCR platform and the digital PCR platform were used to conduct experiments to verify the effect. For each target bacteria, the primer effect was first tested using the Hangzhou Biori Gene 9660 fluorescence quantitative PCR platform (melting curve method), and a single primer pair and an 11-plex primer combination were used for testing. The single primer and the 11-plex primer combination were required to be compared, and the δCt value difference was within ±2 and the δ target peak was within ±250 to be qualified. Then, a single primer pair test was performed using the Pilot fully automatic droplet digital PCR system (AD3207) (probe method; limited to a maximum of 7 multiplexes), and the concentration of drug-resistant gene nucleic acid could be quantitatively obtained. For samples with known concentrations, the difference between the known concentration and the digital PCR determination concentration can be compared, and the δ concentration difference within ±50% is considered qualified. All experimental results are shown in Tables 2-5.

[0151] Table 2-5 Test results of 11-plex primer-probe combination experiment

[0152]

[0153]

[0154] Conclusion: For 11 pathogen targets, single primers and 11-plex primer combinations were first tested using fluorescent quantitative PCR. The results showed that for each primer, the CT value and target peak value of single amplification and combined primer amplification were consistent, indicating that the 11-plex primer combination amplification effect was good; then, the single primer probe sequence was further tested using the digital PCR platform. The results showed that all 11 primer probe sequences could work normally and measure the concentration values ​​of the target pathogens, and the probe quantitative values ​​of Acinetobacter baumannii, Klebsiella pneumoniae, and Pseudomonas aeruginosa using pure bacterial strains as test materials were consistent with the standard concentration values ​​of the strains.

[0155] In summary, the 11-plex primer-probe combinations designed this time targeting pathogens such as Escherichia coli and Klebsiella pneumoniae can work normally.

[0156] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present application, rather than to limit it. Although the present application has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present application.

Claims

1. A method for designing multiple digital PCR primer probes for target pathogens or drug resistance genes, characterized in that: The steps include: 1) Design and select primer and probe sequences for a single target pathogen or a single drug resistance gene; 2) Screen and select primer-probe sequence combinations for all target pathogens or drug-resistant genes; The design and selection in step 1) are: a) Based on all specific core genome sequences of a single target pathogen or a single drug resistance gene template sequence, primers were designed using Primer3 to obtain batch primer pairs and amplicon sequences; b) Use MFEprimer to evaluate and filter the batch primer sequences; for each primer pair's amplicon sequence, screen the probe sequences in other non-primer regions on the amplicon; c) Evaluate the specificity of primer amplification based on the human reference genome sequence, clinical environment-related microbial reference genome sequence, and CARD resistance gene database; d) Use a custom formula to calculate the score of primers / probes and sort the primers / probes from low to high according to the comprehensive score results; The custom formula in step d) is: Among them, Penalty Score is the comprehensive score of the primer probe, F is various factors, and Score is the score of the corresponding factor; Use the defined formula to calculate the score of the primers / probes and sort them according to the score results; use the formula to calculate the comprehensive score of each primer / probe, and sort the primers / probes from low to high according to the comprehensive score value; The scoring rules for the various factors are as follows: The step 2) comprises the following steps: i) For all target pathogens or drug resistance genes, sort them from high to low in terms of clinical importance or from low to high in terms of the number of candidate available primer pairs, and evaluate and gradually expand the combinations of all primer probes to meet the requirements of no secondary structure and non-specific amplification by adding one target primer probe at a time, and screen the primer probe combinations for all targets; ii) Evaluate the effect of each primer / probe pair for the primer / probe combination obtained through screening to ensure that both the primer and the probe have no secondary structure; iii) Design degenerate bases on primers or probes based on template variation information; evaluate the genome coverage of each primer pair for the target pathogen.

2. The method according to claim 1, characterized in that The step b) is specifically as follows: using MFEprimer to evaluate batch primer sequences, filtering primers whose product tm obtained by MFEprimer evaluation differs from primer tm by more than 19°C; filtering primer pairs whose variation frequency on the primer sequence is more than 1% and whose number of variation sites is more than 2 or whose single variation site is at the 3' end according to the nucleotide variation information on the template sequence; for the remaining primer pairs and their amplicons, intercepting 18 to 30 bp sequences from different positions close to the F primer or R primer end, and evaluating whether they can be used as probes; if a qualified probe sequence is found on the amplicon, retaining the current primer, otherwise filtering out the corresponding primer.

3. The method according to claim 2, characterized in that The sequence evaluation of the probe includes: a. sequence base composition: the first base at the 5' end is not G, the sequence does not contain GGGG and AAAAAA, and the GC content is in the range of 30%-80%; b. the sequence tm value is in the range of 64-72°C, and the difference with the primer tm is in the range of 5-10°C; c. the sequence itself has no hairpin structure and dimer structure, and does not form a dimer with the primer; d. the number of sites with a variation frequency >1% in the sequence does not exceed 2.

4. The method according to claim 1, characterized in that: The evaluation in step c) is: For the target pathogen, MFEprimer is used to evaluate the amplification specificity of each primer relative to the human reference genome and clinically relevant microorganisms, and a score is given based on the results.

5. The method according to claim 4, characterized in that The scoring rules are as follows: if human sequences are amplified, 1 point is given, and if the length of the amplicon is less than 300 bp, 2 points are given; if non-specific other pathogens are amplified, 0.25 points are given, and if the length of the amplicon is less than 300 bp, 0.5 points are given; if there is no non-specific amplification, 0 points are given; For drug-resistant genes, the specificity of the target drug-resistant gene relative to other genes was further evaluated, wherein the other genes were other drug-resistant genes in the CARD drug-resistant gene database except the target drug-resistant gene.

6. The method according to claim 1, characterized in that In step 2) i), the method of adding one target primer probe each time is specifically: Starting with the best scoring primer probe for the first target pathogen or resistance gene, the best scoring primer probe for the second target was added to the combination, and the secondary structure and primer amplification specificity of the 2X primer-probe combination were evaluated using MFEprimer software; If there is no secondary structure and non-specific amplification, then add the best primer probe for the third target, otherwise select other candidate primer probes for the second target and add them to the combination for further evaluation; in this way, gradually complete the selection and evaluation of primer probe combinations for all targets; If a suitable primer probe cannot be found for a certain target during the process, the target will be promoted to the first place and the operation will be repeated until the primer probe combinations for all targets that meet the requirements are automatically screened out.

7. The method according to claim 1, characterized in that In the step 2) of ii), the evaluation is performed using Oligo7 software, and in the step 2) of iii), the evaluation is performed using MFEprimer.

8. An electronic device, characterized in that: include: Processor and memory; The processor is connected to a memory, wherein the memory is used to store a computer program, and the processor is used to call the computer program to execute the method according to any one of claims 1 to 7.

9. A computer storage medium, characterized in that: The computer storage medium stores a computer program, wherein the computer program includes program instructions, and when the program instructions are executed by a processor, the method according to any one of claims 1 to 7 is executed.

Images