Single-ended multiplex PCR library building method for pathogenic microorganisms

Abstract

The invention discloses a single-ended multiplex PCR (Polymerase Chain Reaction) library building method for pathogenic microorganisms, relates to the technical field of microbial gene detection, and in particular relates to the single-ended multiplex PCR library building method for the pathogenic microorganisms. The method comprises the following steps: designing primers, downloading a reference genome from NCBI (National Center of Biotechnology Information), sliding to generate a kmer sequence, comparing to an nt reference library by using blast, filtering non-specific kmer, screening proper primer sequences by using MFEprimer, and selecting a pair of inner and outer side primer sequences from each species; pCR amplification is carried out on the basis of the outer side primer by using the inner side primer, the specificity is enhanced, and then the primer is synthesized according to a self-designed primer sequence. After the technical scheme is adopted, the method has the beneficial effects that the pathogenic microorganisms are detected by using an ultra-high-weight PCR technology and a second-generation high-throughput sequencing technology, and a library can be established by adding 10ng of a sample; multiple single-ended primers can be directly mixed for use, and the concentration of the single primers does not need to be adjusted; the RNA sample and the DNA sample can be used for building a database together, so that the experimental operation is simplified.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial gene detection, and in particular to a single-end multiplex PCR library construction method for pathogenic microorganisms. Background Art

[0002] Pathogen detection technologies are mainly divided into traditional culture methods, immunology methods and molecular biology methods. Traditional detection is mainly divided into three categories: smear microscopy, isolation culture and biochemical reaction, tissue cell culture; immunology methods mainly include enzyme-linked immunosorbent assay (ELISA), immunofluorescence method, etc.; genetic detection mainly focuses on nucleic acid molecular hybridization technology, PCR technology and gene chip technology.

[0003] Existing genetic testing methods have certain defects: traditional culture-based detection methods are mainly applicable to bacteria and fungi, the culture cycle of strains is long, it depends on the operator's culture technology, and it is impossible to detect pathogens growing on the culture medium. The culture process is more susceptible to contamination, the identification accuracy of the results is low, and it is easy to cause false positives. Immunological detection of pathogenic microorganisms requires specific reagents and equipment, and cannot detect all pathogens. Methods based on specific primers / probes / antibodies, such as antigen-antibody reactions, PCR reaction detection, and various specific pathogenic microorganisms rapid detection methods all require professional knowledge in the field of microorganisms and cannot achieve high-throughput detection. mNGS technology has low sensitivity for the detection of intracellular bacteria and fungi. For general sample types such as blood, alveolar lavage fluid, sputum, pleural effusion, etc., the host genome contamination ratio is generally above 80%, which leads to very few microbial sequences detected, and a large part of the data cannot be effectively used. For RNA virus samples, since RNA needs to undergo a reverse transcription process and is easily degraded, there is a certain degree of difficulty in detecting RNA viruses. At present, the terminal charges for mNGS single-item tests (DNA or RNA) are relatively expensive. For patients who are not in critical condition or seriously ill, it is generally difficult to receive testing. Summary of the invention

[0004] The purpose of the present invention is to provide a single-end multiplex PCR library construction method for pathogenic microorganisms in view of the defects and shortcomings of the prior art. The method uses single-end multiplex PCR combined with NGS technology to detect pathogenic microorganisms. Single-end multiplex PCR can amplify multiple fragments at the same time, and the library construction of multiple pathogens can be completed within a few hours. The sample starting amount is low, the efficiency is high, and the operation steps are simple. The number of amplicons can reach more than one thousand, and the uniformity is good. Even samples of poor quality can complete the library construction in a short time; single-end specific targeted amplification can avoid the interference of human background to a great extent, reduce sequencing costs, improve detection efficiency, and accurately detect all pathogens in the panel at the same time; four anti-pollution UMI connectors can efficiently monitor the contamination between samples; double-end Index library construction can solve the Index Hopping problem well, thereby realizing accurate sample splitting; and internal reference primers are added to control the experimental process.

[0005] To achieve the above object, the present invention adopts the following technical solution: a single-end multiplex PCR library construction method for pathogenic microorganisms, which comprises the following steps:

[0006] Step 1: Primer design: download the reference genome from NCBI and generate kmer sequences by sliding. Use blast to align to the nt reference library, filter non-specific kmer, use MFEprimer to screen suitable primer sequences, and select a pair of inner and outer primer sequences from each species; use the inner primer to perform PCR amplification based on the outer primer to enhance specificity, and then synthesize primers based on the self-designed primer sequence;

[0007] Step 2: Synthesis of cDNA strand 1: Take out 1st Strand Buffer, thaw and mix by inversion, add 6μL of 1st Strand Buffer, 17μL of RNA and 2μL of 1st Strand Enzyme Mix2 into a PCR tube to prepare a reaction solution, mix by pipetting, centrifuge briefly, place the reaction solution on a PCR instrument, run the program and obtain product A;

[0008] Step 3: cDNA second-strand synthesis, take out the 2nd Strand Buffer and thaw it, mix it by inversion, add 20 μL of the mixed 2nd Strand Buffer, 25 μL of product A, and 5 μL of 2nd Strand Enzyme Mix2 into the PCR tube to prepare a reaction solution, use a pipette to mix, centrifuge it instantly, and then place the reaction solution on the PCR instrument and run the program to obtain product B;

[0009] Step 4: Purification after reverse transcription, take 90 μL of magnetic beads in a tube, add product B, mix well and let stand at room temperature for 10 minutes, then centrifuge briefly, place on a magnetic stand and let stand for 5 minutes, discard the supernatant after the liquid is clear, add 180 μL of freshly prepared 80% ethanol, let stand for 30 seconds, discard the supernatant after the solution is clear, repeat twice, cover the tube cap, centrifuge briefly, centrifuge the residual ethanol to the bottom of the tube, place the eight-tube strip on a magnetic stand, use a 10 μL pipette to remove the residual ethanol, let stand at room temperature for 3 minutes, remove the tube from the magnetic stand, add 53 μL of Nuclease-Free Water, pipette or vortex to mix, let stand at room temperature for 2 minutes, then place the tube on a magnetic stand for 3 minutes, wait for the solution to clarify, aspirate 50 μL of supernatant, transfer to a new PCR tube or eight-tube strip to obtain a mixture of DNA and cDNA;

[0010] Step 5: Fragmentation: Take out FEA Enzyme Mix V3, thaw and mix, centrifuge briefly to collect the reaction solution to the bottom of the tube, and place on ice for later use. Place the sterilized PCR tube on ice, take 10 μL of FEA Enzyme Mix V3 and the mixture of DNA and cDNA into the sterilized PCR tube, mix and centrifuge, collect the reaction solution to the bottom of the tube, and then place the sterilized PCR tube in the PCR instrument and run the program to obtain product C.

[0011] Step 6: Connector ligation. Take out Rapid Ligation Buffer V3 and Rapid DNA Ligase V3 from tubes, thaw and mix well. Centrifuge briefly to collect the solution to the bottom of the tube and place on ice for later use. Place the PCR tube on ice, take 30μL Rapid Ligation Buffer V3, 5μL Rapid DNA Ligase V3, 5μL Adapter and product C into the PCR tube, mix well, centrifuge to collect the reaction solution to the bottom of the tube, then place the sterilized PCR tube in a PCR instrument and run the program to obtain product D.

[0012] Step 7: Purification after connection, take 80μL magnetic beads in an eight-tube strip, add product D, mix well, let stand at room temperature for 5 minutes, centrifuge briefly, place on a magnetic stand and let stand for 2-6 minutes, discard the supernatant after the liquid is clear, add 180μL freshly prepared 80% ethanol, let stand for 30s, discard the supernatant after the solution is clear, repeat twice, cover the tube cap, centrifuge briefly, centrifuge the residual ethanol to the bottom of the tube, place the eight-tube strip on a magnetic stand, use a 10μL pipette to remove the residual ethanol, let stand at room temperature for 3 minutes, then remove the tube from the magnetic stand, add 22μL Nuclease-Free Water, pipette or vortex to mix, let stand at room temperature for 2 minutes, then place the tube on a magnetic stand for 3 minutes, wait for the solution to clarify, aspirate 20μL supernatant, transfer to a new PCR tube or eight-tube strip, and obtain 20μL DNA;

[0013] Step 8: Specific amplification, thaw the required reagents at room temperature, mix them upside down for later use, place the PCR tube on ice, add 20 μL of DNA, 2 μL of specific primers, 2 μL of universal primers, and 6 μL of 5×VAHTS RT Multi-PCRMix to the tube, mix well and centrifuge instantly, place the PCR tube on the PCR instrument, run the program, and obtain product E;

[0014] Step 9: Purification after specific amplification, take 50μL magnetic beads in an eight-tube strip, add all product E, mix well, let stand at room temperature for 5 minutes, centrifuge briefly, place on a magnetic stand and let stand for 2-6 minutes, discard the supernatant after the liquid is clear, add 180μL freshly prepared 80% ethanol, let stand for 30 seconds, discard the supernatant after the solution is clear, repeat twice, use a 10μL pipette to remove the residual ethanol, let stand at room temperature for 3 minutes, add 20μL Nuclease-Free Water, remove the eight-tube strip from the magnetic stand, pipette or vortex to mix, let stand at room temperature for 2 minutes, centrifuge briefly, place the eight-tube strip on the magnetic stand again, wait for the solution to be clear, aspirate 16μL supernatant, transfer to a new PCR tube or eight-tube strip, and mark it as product F;

[0015] Step 10: Post-PCR amplification, place the PCR tube on ice, add product F, 4μ Lindex, and 20μL VAHTS HiFi Amplification Mix into the PCR tube, mix well and centrifuge instantly, place the PCR tube on the PCR instrument, run the program, and obtain product M;

[0016] Step 11: Post-PCR amplification and purification, take 36μL magnetic beads in an eight-tube strip, add all product M, mix, let stand at room temperature for 5min, centrifuge briefly, place on a magnetic stand for 3min, discard the supernatant after the liquid is clear, add 180μL freshly prepared 80% ethanol, let stand for 30s, discard the supernatant after the solution is clear, repeat twice, use a 10μL pipette to remove residual ethanol, let stand at room temperature for 3min, add 35μL Nuclease-Free Water, remove the eight-tube strip from the magnetic stand, pipette or vortex to mix, let stand at room temperature for 2min, centrifuge briefly, place the eight-tube strip on the magnetic stand for 2min, wait for the solution to be clear, aspirate 32μL supernatant, transfer to a new 1.5ml tube, and mark it as product N; measure product N and record library concentration and fragment size; sequence by sequencer.

[0017] The 1st Strand Buffer, 2nd Strand Buffer, Rapid Ligation Buffer V3, and Rapid DNA Ligase V3 were all stored at -20°C.

[0018] The program of the PCR instrument in step 2 is: heat cover at 105°C, run at 25°C for 10 minutes, run at 42°C for 10 minutes, run at 70°C for 15 minutes, and finally maintain at 4°C.

[0019] The program of the PCR instrument in step 3 is: no hot cover, run at 16°C for 60 minutes, and finally keep at 4°C.

[0020] The program of the PCR instrument in step 5 is: heat cover at 105°C, run at 30°C for 5 minutes, run at 72°C for 5 minutes, and finally maintain at 4°C.

[0021] The program of the PCR instrument in step six is: no hot cover, run at 20°C for 15 minutes, and finally keep at 4°C.

[0022] The program of the PCR instrument in step seven is: hot cover at 105°C, run at 95°C for 3 minutes, run at 95°C for 30 seconds, run at 60°C for 1 minute, run at 72°C for 1 minute, run at 72°C for 3 minutes, and finally keep at 4°C.

[0023] The program of the PCR instrument in step ten is: hot cover at 105°C, run at 98°C for 3 minutes, run at 98°C for 30 seconds, run at 60°C for 1 minute, run at 72°C for 30 seconds, run at 72°C for 1 minute, and finally keep at 4°C.

[0024] Working principle of the present invention: This technology uses ultra-multiplex single-end PCR in conjunction with NGS sequencing to build a library for DNA and RNA samples together. It is a tNGS system designed for pathogenic microorganisms. This technology first adds 4 types of UMI connectors to the ends of different fragmented samples (see the UMI information table for sequence information). The UMI connector can identify each DNA molecule to avoid erroneous amplification during PCR and sequencing. At the same time, the offline data can be filtered through bioinformatics analysis. Compared with traditional random UMI, the sequencing results obtained are more accurate; secondly, two rounds of specific PCR amplification are performed on the target area. In the first round of amplification, the target area of ​​the connection product is specifically enriched by the outer primer and the universal primer to obtain the first round of PCR products. The second round of specific PCR amplification is performed by inner primer. r primers and universal primers react and enrich the target area of ​​the first round of products. This technology can effectively detect a variety of pathogenic microorganisms by performing two rounds of specific PCR on the samples and then sequencing them on the machine, and has good specificity. This technology is applied to NGS sequencing of pathogenic microorganisms for the first time; 5% of human internal reference genes are added to the outer primer and inner primer. The microbial content in different samples is different, and the expression level of the internal reference gene in each sample is not affected. The stably expressed human VCP and GPI genes are selected as internal references to normalize the data, ensure that the experimental process is controllable, and ensure that the sequencing results are accurate and reliable.

[0025] After adopting the above technical solution, the beneficial effects of the present invention are:

[0026] 1. Use ultra-high-density PCR technology and second-generation high-throughput sequencing technology to detect pathogenic microorganisms, and 10ng of sample can be used to build a library;

[0027] 2. Multiple single-end primers can be directly mixed and used without adjusting the concentration of single primers;

[0028] 3. RNA samples and DNA samples can be jointly library-built to simplify experimental operations;

[0029] 4. The experiment is easy to operate and the whole process can be completed within 7 hours;

[0030] 5. 4 types of anti-contamination UMI adapters, which remove exogenous DNA sequences and can effectively monitor the contamination between samples;

[0031] 6. The use of single-end multiplex PCR is less likely to produce aerosol contamination, reducing the probability of false positive test results;

[0032] 7. Double-end indexes can be used for orthogonal combinations between indexes, enriching index diversity, increasing the number of final mixed libraries in each lane, effectively reducing the index hopping effect, accurately splitting data, and improving sequencing accuracy;

[0033] 8. There is a complete experimental system and the whole process is controllable. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0035] Figure 1 This is the IGV screenshot of YF20231027T01 in the present invention.

[0036] Figure 2 It is an IGV screenshot of YF20231027T03 in the present invention.

[0037] Figure 3 It is a flow chart related to the present invention. DETAILED DESCRIPTION

[0038] See also Figure 1-3 As shown, the technical solution adopted in this specific implementation is:

[0039] Download high-quality reference genomes of SARS-CoV-2, Klebsiella pneumoniae, Enterococcus faecalis, Staphylococcus aureus, and human housekeeping gene VCP and GPI sequences from NCBI. Slide kmers of 25 bp length from different microorganisms and classify all kmers by species.

[0040] Use blast to align to the nt library and remove kmers that align to species other than SARS-CoV-2, Klebsiella pneumoniae, Enterococcus faecalis, and Staphylococcus aureus; human housekeeping gene primers remove kmers that align to genes other than human VCP and GPI genes.

[0041] Use MFEprimer to evaluate the remaining kmer sequences, and select suitable primer sequences based on the GC content, hairpin structure prediction, dimer structure prediction, etc. calculated by the software. The inner primers are used for PCR amplification based on the outer primers to enhance specificity. Provide self-designed primer sequences and synthesize primers.

[0042] For cDNA one-strand synthesis, take out the 1st Strand Buffer, thaw it and mix it by inversion. Add 6μL of the mixed 1st Strand Buffer, 17μL of RNA, and 2μL of 1st Strand Enzyme Mix2 to the PCR tube to prepare the reaction solution. Use a pipette to mix (avoid vigorous shaking and mixing), and centrifuge it instantly. Then place the reaction solution on the PCR instrument, with a reaction system of 25μL, run the program, and obtain product A;

[0043] cDNA second-strand synthesis: After thawing, take out the 2nd Strand Buffer and mix it by inversion. Add 20 μL of the mixed 2nd Strand Buffer, 25 μL of product A, and 5 μL of 2nd Strand Enzyme Mix2 to the PCR tube to prepare a reaction solution. Use a pipette to mix (avoid vigorous shaking), centrifuge instantly, and then place the reaction solution on the PCR instrument. The reaction system is 50 μL. Run the program to obtain product B.

[0044] After reverse transcription, purify by taking 90 μL of magnetic beads in a tube, adding product B, pipetting or vortexing to mix, letting stand at room temperature for 10 min, centrifuging briefly, placing on a magnetic stand and letting stand for 5 min, discarding the supernatant after the liquid is clear, avoiding touching the magnetic beads, and then adding 180 μL of freshly prepared 80% ethanol, letting stand for 30 s, discarding the supernatant after the solution is clear, repeating twice, covering the tube, centrifuging briefly, centrifuging the residual ethanol to the bottom of the tube, placing the eight-tube strip on a magnetic stand, using a 10 μL pipette to remove the residual ethanol, letting stand at room temperature for 3 min, removing the tube from the magnetic stand, adding 53 μL of Nuclease-Free Water, pipetting or vortexing to mix, letting stand at room temperature for 2 min, and then placing the tube on a magnetic stand for 3 min, waiting for the solution to be clear, pipetting 50 μL of supernatant, transferring it to a new PCR tube or eight-tube strip to obtain a mixture of DNA and cDNA;

[0045] Fragmentation: Take out FEA Enzyme Mix V3, thaw and mix, briefly centrifuge to collect the reaction solution to the bottom of the tube, and place on ice for later use. Place the sterile PCR tube on ice, take 10 μL of FEA Enzyme Mix V3 and the mixture of DNA and cDNA into the sterile PCR tube, mix and centrifuge, collect the reaction solution to the bottom of the tube, and then place the sterile PCR tube in the PCR instrument and run the program to obtain product C.

[0046] Connector connection, take out Rapid Ligation Buffer V3 and Rapid DNA Ligase V3 from the tubes respectively, thaw and mix, centrifuge briefly to collect to the bottom of the tube, put on ice for later use, put the PCR tube on ice, take 30μL RapidLigation Buffer V3, 5μL Rapid DNA Ligase V3, 5μL Adapter, and product C into the PCR tube, mix well, centrifuge to collect the reaction solution to the bottom of the tube, then put the sterilized PCR tube into the PCR instrument, the reaction system is 25μL, run the program, and obtain product D;

[0047] After connection, purification, take 80μL magnetic beads in an eight-tube strip, add product D, mix well, let stand at room temperature for 5 minutes, centrifuge briefly, place on a magnetic stand and let stand for 2-6 minutes, discard the supernatant after the liquid is clear, add 180μL freshly prepared 80% ethanol, let stand for 30 seconds, discard the supernatant after the solution is clear, repeat twice, cover the tube cap, centrifuge briefly, centrifuge the residual ethanol to the bottom of the tube, place the eight-tube strip on a magnetic stand, use a 10μL pipette to remove the residual ethanol, let stand at room temperature for 3 minutes, then remove the tube from the magnetic stand, add 22μL Nuclease-Free Water, mix by pipetting or vortexing, let stand at room temperature for 2 minutes, then place the tube on a magnetic stand for 3 minutes, wait for the solution to be clear, aspirate 20μL supernatant, transfer to a new PCR tube or eight-tube strip, and obtain 20μL DNA;

[0048] For specific amplification, thaw the required reagents at room temperature, mix them upside down for later use, place the PCR tube on ice, add 20 μL of DNA, 2 μL of specific primers, 2 μL of universal primers, and 6 μL of 5×VAHTS RT Multi-PCR Mix to the tube, mix well, centrifuge instantly, place the PCR tube on a PCR instrument, run the program, and obtain product E;

[0049] Purify after specific amplification, take 50μL magnetic beads in an eight-tube strip, add all product E, mix well, let stand at room temperature for 5 minutes, centrifuge briefly, place on a magnetic stand and let stand for 2-6 minutes, discard the supernatant after the liquid is clear, add 180μL freshly prepared 80% ethanol, let stand for 30 seconds, discard the supernatant after the solution is clear, repeat twice, use a 10μL pipette to remove the residual ethanol, let stand at room temperature for 3 minutes, add 20μL Nuclease-Free Water, remove the eight-tube strip from the magnetic stand, pipette or vortex to mix, let stand at room temperature for 2 minutes, centrifuge briefly, place the eight-tube strip on a magnetic stand again, wait for the solution to be clear, aspirate 16μL supernatant, transfer to a new PCR tube or eight-tube strip, and mark it as product F;

[0050] Post-PCR amplification, place the PCR tube on ice, add product F, 4μ Lindex, and 20μL VAHTS HiFi Amplification Mix into the PCR tube, mix well and centrifuge instantly, place the PCR tube on the PCR instrument, run the program, and obtain product M;

[0051] After post-PCR amplification and purification, take 36 μL of magnetic beads in an eight-tube strip, add all the product M, mix well, let stand at room temperature for 5 minutes, centrifuge instantly, place on a magnetic stand and let stand for 3 minutes, discard the supernatant after the liquid is clear, add 180 μL of freshly prepared 80% ethanol, let stand for 30 seconds, discard the supernatant after the solution is clear, repeat twice, use a 10 μL pipette to remove the residual ethanol, let stand at room temperature for 3 minutes, add 35 μL of Nuclease-Free Water, remove the eight-tube strip from the magnetic stand, pipette or vortex to mix, let stand at room temperature for 2 minutes, centrifuge instantly, and then place the eight-tube strip on the magnetic stand for 2 minutes, wait for the solution to be clear, aspirate 32 μL of supernatant, transfer to a new 1.5 ml tube, and mark it as product N; measure product N and record the library concentration and fragment size;

[0052] The product N was sequenced by a sequencing machine, and the sequence of the product N was measured by a sequencing machine. The 1st Strand Buffer, 2nd Strand Buffer, Rapid Ligation Buffer V3, and Rapid DNA Ligase V3 were all stored at -20°C.

[0053] The program of the PCR instrument in step 2 is: heat cover at 105°C, run at 25°C for 10 minutes, run at 42°C for 10 minutes, run at 70°C for 15 minutes, and finally maintain at 4°C.

[0054] The program of the PCR instrument in step 3 is: no hot cover, run at 16°C for 60 minutes, and finally keep at 4°C.

[0055] The program of the PCR instrument in step 5 is: heat cover at 105°C, run at 30°C for 5 minutes, run at 72°C for 5 minutes, and finally maintain at 4°C.

[0056] The program of the PCR instrument in step six is: no hot cover, run at 20°C for 15 minutes, and finally keep at 4°C.

[0057] The program of the PCR instrument in step seven is: hot cover at 105°C, run at 95°C for 3 minutes, run at 95°C for 30 seconds, run at 60°C for 1 minute, run at 72°C for 1 minute, run at 72°C for 3 minutes, and finally keep at 4°C.

[0058] The program of the PCR instrument in step ten is: hot cover at 105°C, run at 98°C for 3 minutes, run at 98°C for 30 seconds, run at 60°C for 1 minute, run at 72°C for 30 seconds, run at 72°C for 1 minute, and finally keep at 4°C.

[0059] Data analysis

[0060] Filter the adapter sequences in the offline data; filter the bases with sequencing quality values ​​less than 20; filter the sequences with sequencing length less than 50bp.

[0061] Unique Molecular Identifier (UMI) analysis.

[0062] Use umi_tools to truncate the UMI labels of the remaining read sequences and place them in the read names. According to the UMI sequences of different samples, filter the read data of the UMI sequence that does not correspond to the sample, and only retain the reads of the specified UMI sequence.

[0063] Human internal reference analysis, retaining microbial data.

[0064] Bowtie2 software was used to align the above-retained reads to the human reference genome (, with default parameters, retaining the sequences that were aligned and those that were not aligned to the human reference genome, respectively.

[0065] For the aligned sequences, picard markers and duplicate removal were used to compare the results, and bedtools was used to count the number of reads in the human internal reference region before and after deduplication, which was the internal reference analysis result. c) For the unaligned sequences, the software hisat2 was used to align them to human mRNA and rRNA, with default parameters, and the unaligned sequences were retained again for subsequent microbial identification.

[0066] Microbial sequence alignment and identification.

[0067] Download high-quality reference genomes such as the new coronavirus from NCBI. Then use bowtie2-build to build a comparison index for different microbial reference genomes.

[0068] Bowtie2 was used to align the unaligned sequences to the established microbial reference genome. Picard markers and deduplication were then used to compare the results. Bedtools was then used to count the number of reads in the microbial target region before and after deduplication.

[0069] The number of reads before and after demultiplexing for different microbial targets was counted separately. If the number of reads detected by the negative reference microorganism is 0, the number of reads detected by the sample to be tested is less than 10, indicating that it is not detected, and the number of reads detected is greater than 10, indicating that it is detected; if the number of reads detected by the negative reference microorganism is not 0, the read detection data of the sample to be tested is greater than 10 times the number of reads detected by the negative reference, indicating that it is detected, otherwise it is not detected.

[0070] UMI analysis can ensure that the reads with UMI sequences are the DNA sequences of the sample. Sequences without specified UMI or without UMI are all exogenous DNA and need to be filtered. The specific results are shown in the following table. Through the corresponding UMI sequence analysis, we can see that the UMI proportions of different samples vary greatly. Only the specified UMI proportion is high, indicating that the cross-contamination of the experimental process is extremely low.

[0071]

[0072] The results of the internal reference analysis are shown in the following table. The total DNA content is low, the human internal references of other samples are detected normally, and the overall experimental process is normal.

[0073] Experimental operation sample Number of GPI internal reference reads Add human GPI internal reference YF20231027T01 5 Add human GPI internal reference YF20231027T02 33154 Add human GPI internal reference YF20231027T03 44243 Add human GPI internal reference YF20231027T04 48224 Add human GPI internal reference YF20231027T05 81015 Add human GPI internal reference YF20231027T06 11

[0074] In the actual detection of real samples, after data analysis, the read number table obtained is as follows:

[0075]

[0076]

[0077] According to the judgment criteria, the final results are interpreted as shown in the following table. The sample data analysis results are completely consistent with the clinical sample type.

[0078]

[0079] The above description is only used to illustrate the technical solution of the present invention rather than to limit it. Other modifications or equivalent substitutions made to the technical solution of the present invention by ordinary technicians in this field should be included in the scope of the claims of the present invention as long as they do not depart from the spirit and scope of the technical solution of the present invention.

Claims

1. A single-end multiplex PCR library construction method for pathogenic microorganisms, characterized in that: It includes the following steps: Step 1: Primer design: download the reference genome from NCBI and generate kmer sequences by sliding. Use blast to align to the nt reference library, filter non-specific kmer, use MFEprimer to screen suitable primer sequences, and select a pair of inner and outer primer sequences from each species; use the inner primer to perform PCR amplification based on the outer primer to enhance specificity, and then synthesize primers based on the self-designed primer sequence; Step 2: Synthesis of cDNA strand 1: Take out 1st Strand Buffer, thaw and mix by inversion, add 6μL of 1st Strand Buffer, 17μL of RNA and 2μL of 1st Strand Enzyme Mix2 into a PCR tube to prepare a reaction solution, mix by pipetting, centrifuge briefly, place the reaction solution on a PCR instrument, run the program and obtain product A; Step 3: cDNA second-strand synthesis, take out the 2nd Strand Buffer and thaw it, mix it by inversion, add 20 μL of the mixed 2nd Strand Buffer, 25 μL of product A, and 5 μL of 2nd Strand Enzyme Mix2 into the PCR tube to prepare a reaction solution, use a pipette to mix, centrifuge it instantly, and then place the reaction solution on the PCR instrument and run the program to obtain product B; Step 4: Purification after reverse transcription, take 90 μL of magnetic beads in a tube, add product B, mix well and let stand at room temperature for 10 minutes, then centrifuge briefly, place on a magnetic stand and let stand for 5 minutes, discard the supernatant after the liquid is clear, add 180 μL of freshly prepared 80% ethanol, let stand for 30 seconds, discard the supernatant after the solution is clear, repeat twice, cover the tube cap, centrifuge briefly, centrifuge the residual ethanol to the bottom of the tube, place the eight-tube strip on a magnetic stand, use a 10 μL pipette to remove the residual ethanol, let stand at room temperature for 3 minutes, remove the tube from the magnetic stand, add 53 μL of Nuclease-Free Water, pipette or vortex to mix, let stand at room temperature for 2 minutes, then place the tube on a magnetic stand for 3 minutes, wait for the solution to clarify, aspirate 50 μL of supernatant, transfer to a new PCR tube or eight-tube strip to obtain a mixture of DNA and cDNA; Step 5: Fragmentation: Take out FEA Enzyme Mix V3, thaw and mix, briefly centrifuge to collect the reaction solution to the bottom of the tube, and place on ice for later use. Place the sterilized PCR tube on ice, take 10 μL of FEA Enzyme Mix V3 and the mixture of DNA and cDNA into the sterilized PCR tube, mix and centrifuge, collect the reaction solution to the bottom of the tube, and then place the sterilized PCR tube in the PCR instrument and run the program to obtain product C. Step 6: Connector ligation. Take out Rapid Ligation Buffer V3 and Rapid DNA Ligase V3 from tubes, thaw and mix well. Centrifuge briefly to collect the solution to the bottom of the tube and place on ice for later use. Place the PCR tube on ice, take 30μL Rapid Ligation Buffer V3, 5μL Rapid DNA Ligase V3, 5μL Adapter and product C into the PCR tube, mix well, centrifuge to collect the reaction solution to the bottom of the tube, then place the sterilized PCR tube in a PCR instrument and run the program to obtain product D. Step 7: Purification after connection, take 80μL magnetic beads in an eight-tube strip, add product D, mix well, let stand at room temperature for 5 minutes, centrifuge briefly, place on a magnetic stand and let stand for 2-6 minutes, discard the supernatant after the liquid is clear, add 180μL freshly prepared 80% ethanol, let stand for 30s, discard the supernatant after the solution is clear, repeat twice, cover the tube cap, centrifuge briefly, centrifuge the residual ethanol to the bottom of the tube, place the eight-tube strip on a magnetic stand, use a 10μL pipette to remove the residual ethanol, let stand at room temperature for 3 minutes, then remove the tube from the magnetic stand, add 22μL Nuclease-Free Water, pipette or vortex to mix, let stand at room temperature for 2 minutes, then place the tube on a magnetic stand for 3 minutes, wait for the solution to clarify, aspirate 20μL supernatant, transfer to a new PCR tube or eight-tube strip, and obtain 20μL DNA; Step 8: Specific amplification, thaw the required reagents at room temperature, mix them upside down for later use, place the PCR tube on ice, add 20 μL of DNA, 2 μL of specific primers, 2 μL of universal primers, and 6 μL of 5×VAHTS RT Multi-PCR Mix to the tube, mix well and centrifuge instantly, place the PCR tube on a PCR instrument, run the program, and obtain product E; Step 9: Purification after specific amplification, take 50μL magnetic beads in an eight-tube strip, add all product E, mix well, let stand at room temperature for 5 minutes, centrifuge briefly, place on a magnetic stand and let stand for 2-6 minutes, discard the supernatant after the liquid is clear, add 180μL freshly prepared 80% ethanol, let stand for 30 seconds, discard the supernatant after the solution is clear, repeat twice, use a 10μL pipette to remove the residual ethanol, let stand at room temperature for 3 minutes, add 20μL Nuclease-Free Water, remove the eight-tube strip from the magnetic stand, pipette or vortex to mix, let stand at room temperature for 2 minutes, centrifuge briefly, place the eight-tube strip on the magnetic stand again, wait for the solution to be clear, aspirate 16μL supernatant, transfer to a new PCR tube or eight-tube strip, and mark it as product F; Step 10: Post-PCR amplification, place the PCR tube on ice, add product F, 4μ Lindex, and 20μL VAHTS HiFi Amplification Mix into the PCR tube, mix well and centrifuge instantly, place the PCR tube on the PCR instrument, run the program, and obtain product M; Step 11: Post-PCR amplification and purification, take 36μL magnetic beads in an eight-tube strip, add all product M, mix, let stand at room temperature for 5min, centrifuge briefly, place on a magnetic stand for 3min, discard the supernatant after the liquid is clear, add 180μL freshly prepared 80% ethanol, let stand for 30s, discard the supernatant after the solution is clear, repeat twice, use a 10μL pipette to remove residual ethanol, let stand at room temperature for 3min, add 35μL Nuclease-Free Water, remove the eight-tube strip from the magnetic stand, pipette or vortex to mix, let stand at room temperature for 2min, centrifuge briefly, place the eight-tube strip on the magnetic stand for 2min, wait for the solution to be clear, aspirate 32μL supernatant, transfer to a new 1.5ml tube, and mark it as product N; measure product N and record library concentration and fragment size; sequence by sequencer.

2. A single-end multiplex PCR library construction method for pathogenic microorganisms according to claim 1, characterized in that: The 1st Strand Buffer, 2nd Strand Buffer, Rapid Ligation Buffer V3, and Rapid DNA Ligase V3 were all stored at -20°C.

3. A single-end multiplex PCR library construction method for pathogenic microorganisms according to claim 1, characterized in that: The program of the PCR instrument in step 2 is: heat cover at 105°C, run at 25°C for 10 min, run at 42°C for 10 min, run at 70°C for 15 min, and finally maintain at 4°C.

4. A single-end multiplex PCR library construction method for pathogenic microorganisms according to claim 1, characterized in that: The program of the PCR instrument in step 3 is: no hot cover, run at 16°C for 60 minutes, and finally keep at 4°C.

5. A single-end multiplex PCR library construction method for pathogenic microorganisms according to claim 1, characterized in that: The program of the PCR instrument in step 5 is: heat cover at 105°C, run at 30°C for 5 minutes, run at 72°C for 5 minutes, and finally maintain at 4°C.

6. A single-end multiplex PCR library construction method for pathogenic microorganisms according to claim 1, characterized in that: The program of the PCR instrument in step six is: no hot cover, run at 20°C for 15 minutes, and finally keep at 4°C.

7. A single-end multiplex PCR library construction method for pathogenic microorganisms according to claim 1, characterized in that: The program of the PCR instrument in step seven is: hot cover at 105°C, run at 95°C for 3 minutes, run at 95°C for 30 seconds, run at 60°C for 1 minute, run at 72°C for 1 minute, run at 72°C for 3 minutes, and finally keep at 4°C.

8. A single-end multiplex PCR library construction method for pathogenic microorganisms according to claim 1, characterized in that: The program of the PCR instrument in step ten is: hot cover at 105°C, run at 98°C for 3 minutes, run at 98°C for 30 seconds, run at 60°C for 1 minute, run at 72°C for 30 seconds, run at 72°C for 1 minute, and finally keep at 4°C.

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