CN116732151A - Target detection semi-quantitative method of multiple PCR system - Google Patents
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Abstract
The invention belongs to the technical field of nucleic acid molecular biology detection, and particularly relates to a target detection semi-quantitative method of a multiplex PCR system. The semi-quantitative method comprises the steps of firstly constructing a correction coefficient of an internal reference, and then obtaining an amplification difference coefficient between the internal reference and a target; adding an internal reference and a sample to be detected into a multiplex PCR system to amplify together, and obtaining a sequencing library after two rounds of multiplex PCR amplification; high-throughput sequencing is carried out on the obtained library, and original sequencing data are obtained: and analyzing the original sequencing data, calculating the concentration of the pathogen target, and evaluating the actual load of the pathogen. The semi-quantitative method adds an internal reference sequence with known content into the PCR amplification of NGS, and carries out two rounds of PCR amplification on the sequence and nucleic acid extracted from respiratory tract samples together to complete library construction, on-machine sequencing and bioinformatics analysis, thereby evaluating the actual content of pathogenic microorganisms in respiratory tract samples to be tested.
Description
Technical Field
The invention belongs to the technical field of nucleic acid molecular biology detection, and particularly relates to a target detection semi-quantitative method of a multiplex PCR system.
Background
Since the development of second generation sequencing (NGS) technology in 2005, the application of second generation sequencing technology in the detection of clinically pathogenic microorganisms is increasing. In recent decades, due to the continuous development of the second generation sequencing technology, the domestic sequencer is continuously improved, a large number of molecular diagnostic products based on the second generation sequencing are continuously developed, and the rapid, comprehensive and accurate characteristics of the molecular diagnostic products provide great help for etiology diagnosis of clinical infection patients.
With the increase of clinical demands, pathogen detection based on second-generation sequencing has also evolved from qualitative detection only to current quantitative detection. Absolute quantification of pathogens based on real-time fluorescent quantitative polymerase chain reaction (Quantitative real-time PCR, qPCR) technology is the classical practice of current pathogen quantification, but cannot be used in large scale in pathogen quantification products due to the limitations of its technical principle and high price. In 16S and metagenome sequencing research, the number and structure of the flora are mainly represented by relative abundance, but because 16S and metagenome sequencing is easily influenced by host content, pathogen concentration, pathogen genome size and sequencing depth, the number of detected sequences hardly reflects the actual content of pathogen, and for judging the reliability of detection and monitoring pathogen load, clinicians cannot master the actual pathogen content in samples to adjust the treatment scheme.
In order to rapidly and accurately quantify the content of a specific pathogenic microorganism in a sample to be tested, a large number of techniques for quantifying pathogenic microorganisms based on NGS methods have been developed on the market. Currently, there are related literature (Tettamanti Boshier F A, srinivasan S, et al, modeling 16S rRNA Gene Amplicon Sequencing with Total Bacterial Load To Infer Absolute Species Concentrations in the Vaginal Microbiome[J, mSystems,5 (2), e 00777-19.) reporting that the relative abundance of species can be obtained by 16S amplicon sequencing and combined with bacterial 16S universal primer qPCR to give the total bacterial load in the sample, from which the relative abundance of flora species in the sample can be deduced, but this method is complex and time consuming to operate and does not allow absolute quantification of individual strains. In addition, there are also reports in the literature (Blauwkamp T A, thair S, rosen M J, et al analytical and clinical validation of amicrobial cell-free DNA sequencing test for infectious disease [ J ]. Nature microbiology,2019,4 (4): 663.) that degenerate base sequences are added as internal control substances in plasma metagenome sequencing, and the content of a pathogen molecule to be detected is estimated based on the detection result of the internal control substances, but the estimated pathogen content is not consistent with the clinically general concentration calculation mode, and the conversion relationship therein is unknown, and guidance cannot be provided in the clinical use. Compared with qPCR technology, the method for capturing the target sequence by multiplex PCR amplification has the remarkable advantages of simple operation, low cost, short-time and large-scale enrichment of the target DNA sequence, and the like, and greatly shortens the detection flow and the detection time. Therefore, a method based on a multiplex PCR (polymerase chain reaction) capture technology and capable of rapidly, accurately and once detecting and quantifying the copy number of the strain in the sample is developed, and the method has great market application value.
Disclosure of Invention
In order to overcome the defects in the prior art, the technical problem to be solved by the invention is to provide a targeted detection semi-quantitative method of a multiplex PCR system, which can rapidly, accurately and comprehensively quantify the copy number of specific pathogenic microorganisms in respiratory tract samples.
In order to solve the technical problems, the technical scheme adopted by the invention is a targeted detection semi-quantitative method of a multiplex PCR system, which comprises the following steps:
s1: constructing correction coefficients of internal parameters;
s2: obtaining an amplification difference coefficient between the internal reference and the target;
s3: adding an internal reference and a sample to be detected into a multiplex PCR system to amplify together, and obtaining a sequencing library after two rounds of multiplex PCR amplification;
s4: carrying out high-throughput sequencing on the obtained library to obtain original sequencing data;
s5: and analyzing the original sequencing data, calculating the concentration of the pathogen target, and evaluating the actual load of the pathogen.
The method has the beneficial effects that the method for quantitatively detecting pathogenic microorganisms in the respiratory tract sample is established based on the NGS sequencing principle combined with the multiple PCR amplification technology. Adding an internal reference sequence with known content into the first PCR amplification of NGS, carrying out two-round PCR amplification on the sequence and nucleic acid extracted from a respiratory tract sample together to complete library construction, on-machine sequencing and bioinformatics analysis, and evaluating the real content of pathogenic microorganisms in the respiratory tract sample to be tested by combining detection conditions of the internal reference and the pathogenic with a theoretical model. The method has the advantages of simplicity in operation, convenience, rapidness, high accuracy, high sensitivity and the like:
(1) The operation is simple: the whole library construction process can be completed only by two rounds of PCR reaction, and other operations are not needed;
(2) Convenient and rapid: compared to mNGS, tNGS only requires 13h to receive a report from a sample;
(3) High accuracy: the internal reference primer and the target pathogen specific primer are amplified and pooled to obtain an internal reference sequence and a target pathogen sequence, the real load of pathogenic microorganisms in the respiratory tract sample is calculated, the internal reference primer and the target pathogen specific primer are not influenced by human background nucleic acid, and a more real and accurate result is provided for NGS sequencing of pathogenic nucleic acid in the respiratory tract sample;
(4) High sensitivity: the limit of quantification can reach 100copies/mL.
Drawings
FIG. 1 is a schematic flow chart of a targeted detection semi-quantitative method of the present invention;
FIG. 2 is a graph showing the relationship between the theoretical concentration and the actual concentration of a part of pathogenic microorganisms in the fifth embodiment of the present invention;
FIG. 3 is a graph showing the relationship between the theoretical concentration and the actual concentration of pathogenic microorganisms in another part of the fifth embodiment of the present invention;
fig. 4 is a plot of the estimated concentration of pathogen in the original sample versus qPCR results for example six of the present invention.
Detailed Description
In order to describe the technical contents, the achieved objects and effects of the present invention in detail, the following description will be made with reference to the embodiments in conjunction with the accompanying drawings.
The most critical concept of the invention is as follows: adding an internal reference sequence with known content into the PCR amplification of NGS, carrying out two rounds of PCR amplification on the sequence and nucleic acid extracted from a respiratory tract sample together to complete library construction, on-machine sequencing and bioinformatics analysis, and evaluating the actual content of pathogenic microorganisms in the respiratory tract sample to be tested.
Referring to fig. 1, the method for semi-quantifying targeted detection of a multiplex PCR system according to the present invention comprises the following steps:
s1: constructing correction coefficients of internal parameters;
s2: obtaining an amplification difference coefficient between the internal reference and the target;
s3: adding an internal reference and a sample to be detected into a multiplex PCR system to amplify together, and obtaining a sequencing library after two rounds of multiplex PCR amplification;
s4: carrying out high-throughput sequencing on the obtained library to obtain original sequencing data;
s5: and analyzing the original sequencing data, calculating the concentration of the pathogen target, and evaluating the actual load of the pathogen.
From the above description, the beneficial effects of the invention are as follows: the invention provides a target detection semi-quantitative method of a multiplex PCR system. The method comprises the following steps: (1) constructing correction coefficients of internal parameters: 3 kinds of internal references for quantification are screened out, the optimal internal reference concentration for quantification is determined, and the weighting coefficients among the 3 kinds of internal references are defined and mutually corrected, so that the internal references are detected more stably and uniformly, which is the basis of the internal references for quantification. (2) obtaining an amplification difference coefficient between the reference and the target: and determining the amplification efficiency difference between the inner reference primer pair and the target primer pair, and avoiding quantitative deviation caused by the amplification efficiency difference of the inner reference primer pair and the target primer pair. (3) The method determines the extraction efficiency value from the original sample to the nucleic acid template, wherein the extraction efficiency value determined by the method is determined by synthesizing the extraction efficiency of each pathogen type under a specific kit. (4) Adding internal reference and a respiratory tract sample to be detected into a multiplex PCR system to amplify together, obtaining a sequencing library after two rounds of multiplex PCR amplification, and performing high-throughput sequencing on the obtained library to obtain original sequencing data; the purpose of the second round of PCR amplification is to allow the enriched internal reference and target pathogen nucleic acids to be ligated to the sequencing library adaptors; (5) Analyzing the sequence numbers of internal references and target detection in the sequencing data, calculating the pathogen concentration in the respiratory tract sample to be detected through a model, finally obtaining the specific copy number of the microorganism in each milliliter of respiratory tract sample, and evaluating the real load of the pathogen.
The semi-quantitative method can rapidly quantify the copy number of the specific target in the respiratory tract sample, has high accuracy, and solves the technical problems that the detection sequence number in the detection of the existing pathogenic microorganism hardly reflects the true content of the pathogen and can cause the omission of detection of certain pathogenic nucleic acid.
The type and sequence of the corresponding target in the invention can only be the target contained in the primer pool; however, the present invention does not completely limit the number of targets that can be quantified, and in a specific implementation process, the targets contained in the primer pool can be added or subtracted by itself according to the detection requirements to set the number and types of targets.
Further, S1 includes the following steps:
s11: manufacturing a target primer pool to be detected, and respectively mixing an internal reference primer and a target pathogenic primer according to the equimolar ratio of an upstream primer and a downstream primer to obtain an upstream amplification primer pool and a downstream amplification primer pool;
s12: respectively diluting the internal reference sequence and the target pathogen sequence to different concentration gradients, and respectively mixing the internal reference sequence and the target pathogen sequence with the same concentration gradient to obtain a template combination;
s13: adding an upstream amplification primer pool and a downstream amplification primer pool into the template combination, and respectively performing two rounds of PCR amplification to obtain a sequencing library;
s14: sequencing the sequencing library and analyzing data to obtain reads (read length) of an internal reference;
s15: repeating S11-S14 to obtain the detected reads of the reference IC1, the reference IC2 and the reference IC3 respectively, and calculating the correction coefficient of the reference, wherein the final correction coefficient is shown as formula 1:
wherein R is IC R is the number of reads calculated after internal reference correction IC1 Number of detection sequences of reference IC1 after RPM (data-normalized detection reads), R IC2 For the number of detection sequences of internal reference IC2 after RPM, R IC3 The number of detection sequences of reference IC3 after RPM.
Further, the length of the internal reference sequence is 100-150 bp, the GC content is 40-60%, and the single base ratio is 20-30%.
From the above description, the reference sequence is designed manually, and does not have cross sequences with the human genome and the pathogenic genome, so that the specificity of the reference sequence is ensured. And limiting the fragment length, GC content and base balance to finally generate an internal reference sequence. The generated internal reference full-length sequence is compared with NT database (Nucleotide Sequence Database) for analysis, and the specificity is high.
Further, the copy number concentration of the reference sequence is 10-7 copies/mL. The copy number concentration of the reference sequence is preferably 10-6 copies/mL.
Further, the difference coefficient K is amplified n The formula of (2) is shown in formula (2):
further, the pathogen target concentration R is calculated N The calculation formula is shown in formula 3:
wherein S is N Number of sequences, K, for pathogenic target RPM detection n R is the amplification difference coefficient between each pathogenic target and internal reference IC For the purpose of sampleThe input amount of the internal reference in the method, F is the correction value from the original sample to the nucleic acid template, S IC The number of sequences was detected for the reference RPM.
Further, the formula of F in formula 3 is shown in formula 4:
F=C (strain concentration copies/ml)/C (nucleic acid concentration copies/ml) formula 4.
Further, the sequencing length of high-throughput sequencing in S4 is greater than PE50, and the sequencing depth of high-throughput sequencing is such that greater than 100 reads can be obtained per target.
Further, analyzing the raw sequencing data in S5 includes sequencing data analysis and sequencing data statistics.
From the above description, the analysis of the raw sequencing data is used to analyze all the sequencing data in the sample to be tested, wherein the sample to be tested contains the nucleic acid sequence of the pathogenic microorganism to be tested and contains the internal reference sequence with known content.
Further, sequencing data analysis includes resolution of Barcode, data quality filtering, removal of low quality reads, removal of non-specific amplification reads and primer dimer reads.
Further, sequencing data statistics are used to count internal reference detected RPM values and pathogen specific detected RPM values in the sequencing data described above.
From the above description, the number of sequences matching to the target under allowable tolerance rules (typically 1-2 base matches) in the sequence with RPM value 100000 is specifically detected.
The first embodiment of the invention is as follows:
preparing an internal reference mixed solution template, which comprises the following steps:
step 1: the determined reference species were pooled with different reference concentrations, and the designed reference sequences (IC 1, IC2, IC 3) were sent to the gene synthesis company (see Table 1 for sequences of reference IC1, IC2, IC3 primers), synthesized by the company and constructed on pUC57 vector. And obtaining nucleic acid dry powder returned by a company after amplification culture, diluting the nucleic acid dry powder by using a TE buffer (TE buffer) to obtain an internal reference mother solution, quantifying the internal reference mother solution by using a Qubit4.0 nucleic acid/protein quantitative fluorometer, and diluting the internal reference mother solution to required concentration to obtain internal reference mixed solution templates with different concentrations. The obtained internal reference mixed liquid templates with different concentrations are as follows: 1000copies/uL, 5000copies/uL and 10000copies/uL.
TABLE 1
Examples: if 10000copies/uL of internal reference mixed liquor template is needed, internal reference IC1, IC2 and IC3 mother liquor is required to be diluted to 100000copies/uL, then each internal reference is mixed after taking 100uL, and then 700uL of TE buffer is added to obtain 10000copies/uL of internal reference mixed liquor template. To obtain a 5000copies/uL internal reference mixed solution template, 10000copies/uL internal reference mixed solution is diluted by 2 times. The 5000copies/uL internal reference mixed solution template is obtained by diluting the 5000copies/uL internal reference mixed solution by 5 times. Wherein the mass concentration and copy number concentration conversion formula is shown in formula 5:
where N is the mass concentration and N is the length (including the template sequence length and plasmid vector length).
The second embodiment of the invention is as follows:
preparation of strain and plasmid mimetic samples comprising the steps of:
firstly extracting nucleic acid from strains, quantifying the concentration of the nucleic acid of the extracted strain by using a Qubit4.0 nucleic acid/protein quantitative fluorometer, and then calculating according to a mass concentration copy number concentration conversion formula shown in a formula 5, wherein N is the genome size of each strain, and the genome size of each strain can be obtained by inquiring on NCBI websites. Each plasmid was prepared by reference to the conventional reference plasmid preparation method. The strain varieties are shown in Table 2.
TABLE 2
After obtaining copy number concentration mother liquor of each strain and plasmid, preparing simulated samples with different concentration gradients under a certain human background, wherein the concentration of the simulated samples is different from 100copies/mL to 10000000copies/mL, and each simulated sample comprises 4-5 strains or plasmids.
The third embodiment of the invention is as follows:
confirming the use concentration of the internal reference, and constructing a correction coefficient, comprising the following steps:
step 1: an internal reference mixed liquor template of 1000copies/uL, 5000copies/uL and 10000copies/uL was prepared by the method of example one;
step 2: and respectively adding an amplification primer pool and a template combination into different internal reference mixed liquid templates, and carrying out first-round PCR amplification, wherein the purpose of the first-round PCR amplification is to enrich the internal reference sequences. The first round PCR reaction system was as follows: first round PCR amplification mix 10uL, first round PCR amplification enzyme 2uL, primer pool 5uL, internal reference template combination (containing 3 concentration gradients) 1uL, H 2 O7 uL; a total of 25uL of amplification system. The amplification primer pool comprises an upstream amplification primer pool and a downstream amplification primer pool, and the inner reference primer and the target pathogenic primer are respectively mixed according to the equimolar ratio of the upstream primer and the downstream primer to obtain the upstream amplification primer pool and the downstream amplification primer pool; and respectively diluting the reference sequence and the target pathogen sequence to different concentration gradients, and respectively mixing the reference sequence and the target pathogen sequence with the same concentration gradient to obtain a template combination.
After the amplification system is prepared, the mixture is uniformly mixed by vortex and then subjected to instantaneous centrifugation, and the amplification is carried out in a PCR instrument according to the following procedures: 3min at 95 ℃; (95 ℃ C. 30s;60 ℃ C. 3min;72 ℃ C. 1 min) 25 cycles; 72 ℃ for 1min; maintained at 4 ℃.
Step 3: and (3) purifying and sorting the PCR products of the first round, and removing primer dimers and other non-target fragments. After the first round of PCR amplification products were obtained, they were purified using the Ampure XP magnetic bead purification kit or other equivalent purification kit. The method comprises the following specific steps: (1) taking 1.5mL of a low adsorption centrifuge tube, adding 25uLNF water, 30uL of magnetic beads and 25uL of amplification product, incubating for 5min at room temperature, transferring to a magnetic rack, waiting for about 3min until the magnetic beads are completely adsorbed, transferring supernatant to a new hole, and taking off from the magnetic rack. (2) 30uL of magnetic beads are added to the supernatant of the first step, incubated for 5min at room temperature, transferred to a magnetic rack and waited for about 3min until the magnetic beads are completely adsorbed, and the supernatant is discarded. (3) The centrifuge tube was kept on a magnetic rack and washed 2 times with 200uL of freshly prepared 80% ethanol each time. Adding 80% ethanol, standing on a magnetic rack for 30s, or blowing and sucking for 3-5 times by using a pipettor to discard the waste liquid. (4) And (3) washing, drying until no water trace exists on the surface of the magnetic beads, adding 15uLNF water, incubating for 5min, transferring to a magnetic frame, and taking 10uL of products for carrying out a second round of PCR after the magnetic beads are completely adsorbed.
Step 4: the purpose of the second round of PCR amplification was to allow the enriched internal reference to be ligated to the sequencing library adaptor, the second round of PCR reaction was as follows: second round PCR amplification mix 25uL, second round PCR amplification enzyme 1uL, barcode primer 1uL, H 2 O13 uL, 10uL of the first round PCR purified product. After the reaction system is prepared, vortex mixing and instantaneous centrifugation are carried out. The following procedure was run on a PCR instrument: 95 ℃ for 2min30s; (95 ℃ 30s;62 ℃ 35s;72 ℃ 35 s) 13-17 cycles; 72 ℃ for 5min; maintained at 4 ℃.
Step 5: the second round PCR products were purified. The kit is consistent with the first round PCR purification kit. The purification and separation steps are as follows: (1) taking 1.5mL of a low adsorption centrifuge tube, adding 50uL of magnetic beads and 50uLPCR amplification products, incubating for 5min at room temperature, transferring to a magnetic rack for about 3min until the magnetic beads are completely adsorbed, and discarding the waste liquid. (2) The centrifuge tube was kept on a magnet rack and washed 2 times with freshly prepared 80% ethanol and 2 times with 150uL of freshly prepared 80% ethanol and 200uL each. Adding 80% ethanol, standing on a magnetic rack for 30s, or blowing and sucking for 3-5 times by using a pipettor to discard the waste liquid. (3) After washing, uncovering and drying until no water trace exists on the surface of the magnetic beads for reflecting light, adding 25uL of 0.1 xTE, incubating for 5min, transferring to a magnetic rack, and transferring 23uL of supernatant to a new 1.5mL low-adsorption centrifuge tube after the magnetic beads are completely adsorbed. The product of this round can be stored at-20 ℃ or used directly for experiments.
Step 6: 1uL of library product was taken, 199uL of dye solution was added, and after thorough mixing, incubated for 2min in the dark, and then quantified using a Qubit4.0 nucleic acid/protein quantitative fluorometer.
Step 7: 2uL library product was taken and fragment size analysis was performed using the Agilent 4150 TapeStation system. The 2uL library and the 2uL assay buffer were mixed to prepare a fragment assay working solution, and fragment size analysis was performed using the Agilent 4150 TapeStation system.
Step 8: library preparation and on-press. All libraries were mixed according to a mass 1:1, sequenced using an MGI-200 platform, and the sequencing kit was an MGISEQ-200RS high throughput sequencing kit (FClSE 50) and loaded according to the instructions above.
Step 9: and (5) analyzing the data of the machine. Barcode resolution, data quality filtration, removal of low quality reads, and removal of primer dimer reads. Finally, comparing the templates to determine the reads number of the templates. And counting the number of reads of the internal reference and the proportion of the internal reference in the total machine-setting data.
Step 10: and detecting more than 1000 internal standard sequences according to the requirement that the internal standard sequence accounts for not more than 20%, wherein the optimal quantitative concentration of the finally obtained internal reference is 1000copies/uL.
Step 11: counting the RPM detection sequence number of 3 kinds of internal references, taking the internal reference of the highest detection sequence number as a correction coefficient 1, and taking the correction coefficients of other internal references as the ratio of the highest internal reference detection sequence number to the correction coefficient. The method is characterized in that: the number of the detected sequences of the IC1 is highest, and the correction coefficient is 1; the correction coefficient of IC2 is determined as 2 by dividing the number of detected sequences of IC1 by the number of detected sequences of IC 2; the correction coefficient of IC3 is determined as 2.5 by dividing the number of detected sequences of IC1 by the number of detected sequences of IC 3.
Step 12: the final internal reference correction formula of the method is shown as formula 1:
the fourth embodiment of the invention is as follows:
calculating the amplification difference coefficient K between the internal reference and the target n The method comprises the following steps:
diluting the internal reference sequence and the target pathogen sequence to concentration gradients of 100, 1000, 10000, 100000, 1000000 and 10000000copies/uL respectively, mixing the internal reference and the target with the same concentration gradient to obtain multiple template combinations, and constructing a library according to the third embodiment to obtain the RPM internal standard detection sequenceThe number of columns and the number of RPM pathogen detection sequences of different strains, and then K is determined by the method of 2 n Value, target K obtained n The values are shown in Table 3.
TABLE 3 Table 3
| Target name | K n Value of | Target name | K n Value of |
| Human herpesvirus 3 | 18.65 | Candida tropicalis | 9.60 |
| Mycobacterium tuberculosis | 25.02 | Pseudomonas maltophilia | 29.91 |
| EB virus | 4.05 | Klebsiella oxytoca | 5.72 |
| Serratia marcescens | 94.60 | Streptococcus agalactiae | 25.38 |
| Cryptococcus garteus | 47.03 | Acinetobacter baumannii | 10.30 |
| Proteus intermedia (L. Intermedia) | 24.02 | Streptococcus pneumoniae | 5.47 |
| Human herpesvirus type 1 | 22.79 | Enterococcus faecium | 4.04 |
| Streptococcus pyogenes | 14.57 | Staphylococcus aureus | 10.48 |
| Haemophilus parainfluenza | 47.50 | Candida albicans | 27.79 |
| BK virus | 7.88 | Candida glabrata (L.) C. | 10.38 |
| Escherichia coli | 16.99 | Cryptococcus neoformans | 62.65 |
| Pseudomonas aeruginosa | 9.60 | Aspergillus fumigatus | 82.13 |
| Klebsiella pneumoniae | 1.25 | COVID-19 | 13.12 |
| Staphylococcus epidermidis | 24.59 | Human cytomegalovirus | 35.00 |
The purpose of the different concentration gradients is to obtain templates of different concentrations for determining the amplification difference coefficient K between the reference and the target n 。
The fifth embodiment of the invention is as follows:
a targeted detection semi-quantitative method of a multiplex PCR system comprises the following steps:
s1: the correction coefficient of the internal reference is constructed by adopting the method of the third embodiment, and the obtained correction coefficient is represented by formula 1:
s2: obtaining the amplification difference coefficient K between the internal reference and the target by the method of the fourth embodiment n The obtained K n The values are shown in Table 3.
S3: adding 1000copies of internal reference mixed liquid template and a sample to be detected into a multiplex PCR system to amplify together, and obtaining a sequencing library after two rounds of multiplex PCR amplification, wherein the amplification method and the library construction method are the same as those of the third embodiment.
S4: all libraries are mixed according to the mass of 1:1, and are sequenced by using an MGI-200 platform, a sequencing kit is MGISEQ-200RS high-throughput sequencing kit (FClSE 50), the sequencing is performed on the machine according to the upper machine instruction, the sequencing length of the high-throughput sequencing is longer than PE50, the sequencing depth of the high-throughput sequencing is longer than 100 reads for each target, and the original sequencing data are obtained.
S5: separating Barcode, filtering data quality, removing low quality reads, removing nonspecific amplification reads and sequencing data analysis of primer dimer reads, counting internal reference detected RPM value and pathogen specific detected RPM value in the sequencing data after analysis, and calculating pathogen target concentration R by using 3 N 。
Wherein S is N Number of sequences, K, for pathogenic target RPM detection n R is the difference coefficient of amplification efficiency between each pathogenic target and internal reference IC F is the correction value from the original sample to the nucleic acid template, S IC The number of sequences was detected for the reference RPM. The formula of F is shown in formula 4:
f=c (strain concentration copies/ml)/C (nucleic acid concentration copies/ml) formula 4;
the average value of F for the various pathogens in Table 2 is 6.67, so the value of F in formula 3 is 6.67.
Pathogen target concentration R N The calculation results of (2) are shown in Table 4. Fitting graphs of linear analyses of pathogen concentrations are shown in figures 2 and 3.
As can be seen from fig. 2 and 3: r of each pathogen at different concentration gradients (100 copies/mL-10000000 copies/mL) 2 The repeatability and the accuracy of the quantitative calculation method are good and are all above 0.98.
TABLE 4 Table 4
The sixth embodiment of the invention is:
the semi-quantitative method of the fifth embodiment is subjected to accuracy testing, and comprises the following steps:
9 samples of patients with positive mNGS detection were collected, and pathogen content was within coverage. The whole flow detection comprises sample processing, library establishment, on-machine sequencing and off-machine data analysis, and finally the estimated concentration of the pathogen in the original sample is obtained and compared with the qPCR result. The calculated concentrations of the two are subjected to logarithmic conversion, the best linear fitting is carried out on the logarithm, the fitting result is shown in fig. 4, and as can be seen from fig. 4, the quantitative method disclosed by the invention has a good fitting effect and high accuracy.
In order to verify the accuracy of the quantitative test, the estimated pathogen concentration and the measured qPCR result are subjected to logarithmic transformation, and the concentration obtained by the two methods is found to be more consistent, R 2 0.9365 (see FIG. 4).
In conclusion, the semi-quantitative method can be used for quantitatively analyzing the target pathogens, can clearly give out the number of copies of pathogenic microorganisms in the sample to be tested, and can detect a plurality of target pathogens in a single sample at the same time.
In summary, the present invention provides a targeted detection semi-quantitative method for multiplex PCR systems. The method comprises the following steps: (1) constructing correction coefficients of internal parameters: 3 kinds of internal references for quantification are screened out, the optimal internal reference concentration for quantification is determined, and the weighting coefficients among the 3 kinds of internal references are defined and mutually corrected, so that the internal references are detected more stably and uniformly, which is the basis of the internal references for quantification. (2) obtaining an amplification difference coefficient between the reference and the target: and determining the amplification efficiency difference between the inner reference primer pair and the target primer pair, and avoiding quantitative deviation caused by the amplification efficiency difference of the inner reference primer pair and the target primer pair. (3) The method determines the extraction efficiency value from the original sample to the nucleic acid template, wherein the extraction efficiency value determined by the method is determined by synthesizing the extraction efficiency of each pathogen type under a specific kit. (4) Adding internal reference and a respiratory tract sample to be detected into a multiplex PCR system to amplify together, obtaining a sequencing library after two rounds of multiplex PCR amplification, and performing high-throughput sequencing on the obtained library to obtain original sequencing data; the purpose of the second round of PCR amplification is to allow the enriched internal reference and target pathogen nucleic acids to be ligated to the sequencing library adaptors; (5) Analyzing the sequence numbers of internal references and target detection in the sequencing data, calculating the pathogen concentration in the respiratory tract sample to be detected through a model, finally obtaining the specific copy number of the microorganism in each milliliter of respiratory tract sample, and evaluating the real load of the pathogen.
The foregoing description is only illustrative of the present invention and is not intended to limit the scope of the invention, and all equivalent changes made by the specification and drawings of the present invention, or direct or indirect application in the relevant art, are included in the scope of the present invention.
Claims (10)
1. The targeted detection semi-quantitative method of the multiplex PCR system is characterized by comprising the following steps of:
s1: constructing correction coefficients of internal parameters;
s2: obtaining an amplification difference coefficient between the internal reference and the target;
s3: adding an internal reference and a sample to be detected into a multiplex PCR system to amplify together, and obtaining a sequencing library after two rounds of multiplex PCR amplification;
s4: carrying out high-throughput sequencing on the obtained library to obtain original sequencing data;
s5: and analyzing the original sequencing data, calculating the concentration of the pathogen target, and evaluating the actual load of the pathogen.
2. The targeted detection semi-quantitative method of a multiplex PCR system according to claim 1, wherein S1 comprises the steps of:
s11: manufacturing a target primer pool to be detected, and respectively mixing an internal reference primer and a target pathogenic primer according to the equimolar ratio of an upstream primer and a downstream primer to obtain an upstream amplification primer pool and a downstream amplification primer pool;
s12: respectively diluting the internal reference sequence and the target pathogen sequence to different concentration gradients, and respectively mixing the internal reference sequence and the target pathogen sequence with the same concentration gradient to obtain a template combination;
s13: adding an upstream amplification primer pool and a downstream amplification primer pool into the template combination, and respectively performing two rounds of PCR amplification to obtain a sequencing library;
s14: sequencing the sequencing library and analyzing data to obtain the reads number of the internal reference;
s15: repeating S11-S14 to obtain the detected reads of the reference IC1, the reference IC2 and the reference IC3 respectively, and calculating the correction coefficient of the reference, wherein the final correction coefficient is shown as formula 1:
wherein R is IC R is the number of reads calculated after internal reference correction IC1 R is the number of detection sequences of internal reference IC1 after RPM IC2 For the number of detection sequences of internal reference IC2 after RPM, R IC3 The number of detection sequences of reference IC3 after RPM.
3. The targeted detection semi-quantitative method of the multiplex PCR system according to claim 2, wherein the length of the internal reference sequence is 100-150 bp, the GC content is 40-60%, and the single base ratio is 20-30%.
4. The targeted detection semi-quantitative method of a multiplex PCR system according to claim 2, wherein the copy number concentration of the internal reference sequence is 10-6 copies/mL.
5. The method for semi-quantitative targeted detection of multiplex PCR system according to claim 1, wherein the amplification difference coefficient K n The formula of (2) is shown in formula (2):
6. the method for semi-quantitative targeted detection of multiplex PCR systems according to claim 1, wherein the concentration R of pathogenic target is calculated N The calculation formula is shown in formula 3:
wherein S is N Number of sequences, K, for pathogenic target RPM detection n R is the amplification difference coefficient between each pathogenic target and internal reference IC F is the correction value from the original sample to the nucleic acid template, S IC The number of sequences was detected for the reference RPM.
7. The method for semi-quantitative targeted detection of multiplex PCR system according to claim 6, wherein the formula for calculating F in formula 3 is shown in formula 4:
F=C (strain concentration copies/ml)/C (nucleic acid concentration copies/ml) formula 4.
8. The targeted detection semi-quantitative method of a multiplex PCR system according to claim 1, wherein the sequencing length of the high throughput sequencing in S4 is greater than PE50 and the sequencing depth of the high throughput sequencing is greater than 100 reads per target.
9. The targeted detection semi-quantitative method of a multiplex PCR system according to claim 1, wherein the analysis of raw sequencing data in S5 includes sequencing data analysis and sequencing data statistics.
10. The targeted detection semi-quantitative method of a multiplex PCR system according to claim 9, wherein the sequencing data analysis includes resolution of Barcode, data quality filtering, removal of low quality reads, removal of non-specific amplification reads and primer dimer reads.
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