Amplicon library building kit for detecting multiple pathogens, library building method and primer group

Abstract

The invention discloses an amplicon library building kit for detecting various pathogens, a library building method and a primer group. The kit comprises a first primer group or a second primer group, and one-step library building can be carried out. According to the invention, the amplicon library building kit, the library building method and the primer group for detecting various pathogens, which can reduce aerosol pollution, simplify library building steps and have high accuracy, can be provided.

Description

Technical Field

[0001] The present disclosure relates to the field of the biomedical industry, and particularly relates to an amplicon library construction kit for detecting multiple pathogens, a library construction method, and a primer set. Background Art

[0002] Polymerase chain reaction (i.e., PCR) is a technology widely used in molecular diagnosis, which is used to amplify a specific DNA fragment to a detectable level. Along with the development of sequencing technology, especially high-throughput sequencing technologies such as next-generation sequencing technology (i.e., NGS technology), it is possible to simultaneously amplify and detect multiple DNA fragments, that is, it is possible to amplify multiple DNA fragments through multiplex PCR technology. Different from the traditional PCR reaction system that only performs 3 to 5 multiplexes, at most 15 multiplexes, the high-multiplex PCR (high-multiplex PCR or ultrahigh-multiplex PCR) technology with hundreds or thousands of multiplexes or even tens of thousands of multiplexes supplemented by NGS sequencing, forming the targeted-NGS technology (i.e., tNGS technology), has gradually played a role in the fields of infectious disease screening, genetic disease diagnosis, tumor gene detection, etc. For the detection of multiple pathogens, usually the high-multiplex PCR method is adopted, and primers capable of specifically amplifying multiple pathogens are designed and synthesized to detect the presence and content of multiple pathogens in a sample. The current targeted sequencing kits for multiple pathogens, including the kits based on probe capture library construction method, design a series of probes to hybridize with the gene sequences of multiple pathogens, so as to capture the pathogen genes, but this method has the disadvantages of high starting amount requirement, relatively high detection limit, cumbersome operation, and high cost. In view of the above deficiencies, the amplicon library construction method can play better advantages. The amplicon library construction uses primers to specifically bind to the target gene and amplifies the target gene fragment through high-multiplex PCR.

[0003] The current amplicon library construction kits usually include four kinds of primers. First, the forward specific primer and the reverse specific primer are used to perform high-multiplex PCR amplification on the target gene sequence, and then another set of forward primer and reverse primer are used for the second round of PCR amplification, so as to add sample tags, adapters required by the sequencing platform, and sequencing primers to both ends of the target gene sequence, obtaining a library that can be used for sequencing. The above process is also called amplicon library construction.

[0004] However, in the current amplicon library construction kits of the above mainstream route, during the product purification process after the first round of amplification, the aerosol contamination generated is likely to lead to the risk of false positives; and two rounds of PCR amplification and purification are required, with numerous steps and cumbersome operations. In addition, the two-round PCR amplification has high requirements for laboratory zoning. Summary of the Invention

[0005] The present disclosure is completed in view of the above-mentioned prior art situation, and its purpose is to provide an amplicon library construction kit, a library construction method, and a primer set for detecting multiple pathogens, which can reduce aerosol pollution, simplify the library construction steps, and have high accuracy.

[0006] To this end, a first aspect of the present disclosure provides an amplicon library construction kit for detecting multiple pathogens, characterized in that the kit includes a first primer set or a second primer set. The first primer set includes a first upstream primer, a first downstream primer, and a second downstream primer. The first upstream primer sequentially includes a first sequencing adapter, a first sample tag sequence for identifying different samples, a first sequencing primer, and a first complementary sequence complementary to the 5'-end of the sequences of the multiple pathogens from its 5'-end to 3'-end. The first downstream primer sequentially includes a second sequencing primer and a second complementary sequence complementary to the 3'-end of the sequences of the multiple pathogens from its 5'-end to 3'-end. The second downstream primer sequentially includes a second sequencing adapter, a second sample tag sequence for identifying different samples, and a second sequencing primer from its 5'-end to 3'-end. The second primer set includes a third upstream primer, a fourth upstream primer, and a third downstream primer. The third upstream primer sequentially includes a fourth sequencing primer and a fourth complementary sequence complementary to the 5'-end of the sequences of the multiple pathogens from its 5'-end to 3'-end. The fourth upstream primer sequentially includes a fourth sequencing adapter, a fourth sample tag sequence for identifying different samples, and a fourth sequencing primer from its 5'-end to 3'-end. The third downstream primer sequentially includes a third sequencing adapter, a third sample tag sequence for identifying different samples, a third sequencing primer, and a third complementary sequence complementary to the 3'-end of the sequences of the multiple pathogens from its 5'-end to 3'-end.

[0007] In the amplicon library construction kit of the first aspect of the present disclosure, when the kit includes the first primer set, the first upstream primer, the first downstream primer, and the second downstream primer can be used to perform a round of PCR amplification simultaneously to amplify and construct a library for the pathogen gene sequence. Compared with the two-round library construction, it can simplify the operation steps, reduce the risk of aerosol contamination, and improve the detection efficiency. Specifically, the 5' end of the pathogen gene sequence can be tagged with the first sequencing adapter and the first sequencing primer through the first upstream primer, and the 3' end of the pathogen gene sequence can be tagged with the second sequencing adapter and the second sequencing primer through the first downstream primer and the second downstream primer; moreover, the first upstream primer and the second downstream primer also carry sample tags, which can enable the pathogen gene sequence to carry sample tags for distinguishing different samples. The sample tags are introduced at the initial stage of PCR, thereby reducing cross-contamination between samples, enabling the library to perform dual-index sequencing, and having a wider adaptability; when the kit includes the second primer set, the effect can be similar to that of the first primer set. Thus, an amplicon library construction kit for detecting multiple pathogens that can reduce aerosol contamination, simplify the library construction steps, and have high accuracy can be provided.

[0008] In the amplicon library construction kit according to the first aspect of the present disclosure, optionally, the first sequencing adapter, the first sequencing primer, the second sequencing adapter, the second sequencing primer, the third sequencing adapter, the third sequencing primer, the fourth sequencing adapter, and the fourth sequencing primer are sequencing adapters and sequencing primers of the Illumina sequencing platform, the MGI sequencing platform, or the Ion Torrent sequencing platform.

[0009] In the amplicon library construction kit according to the first aspect of the present disclosure, optionally, the amplicon library construction kit further includes an internal reference sequence that can be complementary to the first upstream primer and the first downstream primer, or to the third upstream primer and the third downstream primer, and the internal reference sequence has a different sequence from the gene sequences of the multiple pathogens. Thus, quantitative detection of the pathogen can be performed.

[0010] In the amplicon library construction kit according to the first aspect of the present disclosure, optionally, the multiple pathogens include Epstein-Barr virus, Klebsiella aerogenes, Streptococcus agalactiae, Mycobacterium tuberculosis complex, Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa, Haemophilus influenzae, Streptococcus pyogenes, Streptococcus pneumoniae, Human herpes simplex virus 1, Cytomegalovirus, Escherichia coli, Enterobacter cloacae, Klebsiella oxytoca, Proteus mirabilis, Enterococcus faecalis, Enterococcus faecium, Burkholderia cepacia complex, Stenotrophomonas maltophilia, Aspergillus, Acinetobacter baumannii, Cryptococcus neoformans, Pneumocystis jirovecii, Serratia marcescens, Listeria monocytogenes, Bacillus cereus group, Bacteroides fragilis, Mucor, Rhizopus, Candida, Lichtheimia, Rhizomucor, Coagulase-negative staphylococci, Enterococcus, Viridans streptococci group, Bacteroides vulgatus, Salmonella enterica, Prevotella, Citrobacter. Thus, these 40 pathogens can be detected by the kit of the present disclosure.

[0011] In the amplicon library construction kit according to the first aspect of the present disclosure, optionally, in the first primer set, the first complementary sequences are SEQ ID NO.7, SEQ ID NO.15, SEQ ID NO.44, SEQ ID NO.45, SEQ ID NO.54, SEQ ID NO.55, SEQ ID NO.82, SEQ ID NO.83, SEQ ID NO.86 to SEQ ID NO.89, SEQ ID NO.100 to SEQ ID NO.109, SEQ ID NO.225 to SEQ ID NO.282, and the second complementary sequences are SEQ ID NO.9, SEQ ID NO.120, SEQ ID NO.149, SEQ ID NO.150, SEQ ID NO.159, SEQ ID NO.160, SEQ ID NO.187, SEQ ID NO.188, SEQ ID NO.191 to SEQ ID NO.194, SEQ ID NO.205 to SEQ ID NO.214, SEQ ID NO.283 to SEQ ID NO.340. In this case, amplification can be carried out efficiently and accurately by primers specifically designed for 40 pathogens.

[0012] In the amplicon library construction kit according to the first aspect of the present disclosure, optionally,The multiple pathogens include Staphylococcus aureus, Mycobacterium tuberculosis complex, Streptococcus pneumoniae, Haemophilus influenzae, Pseudomonas aeruginosa, Klebsiella pneumoniae, Streptococcus pyogenes, Epstein-Barr virus, Influenza A virus H1N1, Influenza B virus, Human herpesvirus 1, Human herpesvirus 2, Human respiratory syncytial virus A, Human respiratory syncytial virus B, Human coronavirus 229E, Human coronavirus NL63, Human coronavirus OC43, Coronavirus HKU1, Mycoplasma pneumoniae, Aspergillus flavus, Varicella-zoster virus, Cytomegalovirus, Enterovirus genus, Enterovirus D68, Human herpesvirus 6, Escherichia coli, Moraxella catarrhalis, Enterovirus group A, Enterovirus group B, Enterovirus group C, Human parvovirus B19, WU polyomavirus, Bordetella holmesii, Influenza C virus, Rhinovirus A, Rhinovirus B, Rhinovirus C, Histoplasma capsulatum, Mucor racemosus, Rhizopus oryzae, Bordetella pertussis, Talaromyces marneffei, Human parechovirus A, Human herpesvirus 7, BK polyomavirus, Enterobacter cloacae, Chlamydia psittaci, Corynebacterium striatum, Serratia marcescens, Mycobacterium scrofulaceum, Mycobacterium fortuitum, Klebsiella aerogenes, Mycobacterium smegmatis, Klebsiella oxytoca, Scedosporium apiospermum, Pasteurella multocida, Tropheryma whipplei, Elizabethkingia meningoseptica, Chlamydia trachomatis, Orientia tsutsugamushi, Burkholderia mallei, Proteus mirabilis, Burkholderia pseudomallei, JC polyomavirus, Rhodococcus equi, Rickettsia rickettsii, Rickettsia prowazekii, Nocardia gilsonii, Nocardia asteroides, Nocardia fossicola, Nocardia brasiliensis, Nocardia farcinica, Cryptococcus gattii complex, Enterococcus faecalis, Rickettsia typhi, Enterococcus faecium, Staphylococcus epidermidis, Staphylococcus haemolyticus, Enterovirus group D, Burkholderia cepacia complex, Stenotrophomonas maltophilia, Aspergillus genus, Streptococcus agalactiae, Salmonella enterica subsp. enterica, Candida albicans, Candida glabrata, Candida auris, Aspergillus fumigatus, KPC, NDM, vanA, vanB, qnrS, Acinetobacter baumannii, mecA, mcr, Macrolides, Middle East respiratory syndrome coronavirus, SARS coronavirus, Coronavirus disease 2019, Omicron variant of SARS-CoV-2, Enterovirus 71, Influenza A virus, Coxiella burnetii, Francisella tularensis, Human parainfluenza virus 1, Human parainfluenza virus 2, Human parainfluenza virus 3, Human parainfluenza virus 4, Human metapneumovirus, Bocavirus genus, Rubella virus, Measles virus, Mycobacterium abscessus, Mycobacterium kansasii, Cryptococcus neoformans, Pneumocystis jirovecii, Chlamydophila pneumoniae, Human adenovirus group B, Human adenovirus group C, Human adenovirus group E, Mumps virus, Bordetella parapertussis, Bordetella bronchiseptica, Legionella pneumophila, Nocardia spp., Mycobacterium xenopi, Mycobacterium intracellulare, Mycobacterium avium, Mycobacterium gordonae, Mycobacterium chelonae, Neisseria meningitidis, Influenza A virus H3N2, Influenza A virus H5N1, and Influenza A virus H7N9. Thus,The kit of the present disclosure can detect these 127 pathogens and 8 drug resistance genes.

[0013] In the amplicon library construction kit according to the first aspect of the present disclosure, optionally, in the second primer set, the third complementary sequence is SEQ ID NO.7, SEQ ID NO.15 to SEQ ID NO.113, SEQ ID NO.225 to SEQ ID NO.250, SEQ ID NO.346 to SEQ ID NO.489, and the fourth complementary sequence is SEQ ID NO.9, SEQ ID NO.120 to SEQ ID NO.218, SEQ ID NO.283 to SEQ ID NO.308, SEQ ID NO.490 to SEQ ID NO.633. In this case, primers specifically designed for 127 pathogens and 8 drug resistance genes can perform amplification efficiently and accurately.

[0014] In the amplicon library construction kit according to the first aspect of the present disclosure, optionally, in the second primer set, the fourth upstream primer is phosphorylated. Thereby, the library can be adapted to the MGI sequencing platform.

[0015] The second aspect of the present disclosure provides an amplicon library construction method for detecting multiple pathogens, including the following steps: preparing a nucleic acid sample to be tested, obtaining sample DNA, wherein when the nucleic acid sample to be tested is RNA, reverse transcription is first performed; using the kit according to the first aspect of the present disclosure to perform PCR amplification on the sample DNA; obtaining an amplicon library. In the second aspect of the present disclosure, an amplicon library can be obtained by using a one-step library construction method. Thereby, an amplicon library construction method for detecting multiple pathogens that can reduce aerosol contamination, simplify the library construction steps, and has high accuracy can be provided.

[0016] The third aspect of the present disclosure provides a primer set for constructing an amplicon library for detecting multiple pathogens, characterized in that the primer set has two combination forms, namely combination form one and combination form two. The combination form one includes a first upstream primer, a first downstream primer, and a second downstream primer. The first upstream primer sequentially includes a first sequencing adapter, a first sample tag sequence for identifying different samples, a first sequencing primer, and a first complementary sequence complementary to the 5'-end of the sequences of the multiple pathogens from its 5'-end to 3'-end. The first downstream primer sequentially includes a second sequencing primer and a second complementary sequence complementary to the 3'-end of the sequences of the multiple pathogens from its 5'-end to 3'-end. The second downstream primer sequentially includes a second sequencing adapter, a second sample tag sequence for identifying different samples, and a second sequencing primer from its 5'-end to 3'-end. The combination form two includes a third upstream primer, a fourth upstream primer, and a third downstream primer. The third upstream primer sequentially includes a fourth sequencing primer and a fourth complementary sequence complementary to the 5'-end of the sequences of the multiple pathogens from its 5'-end to 3'-end. The fourth upstream primer sequentially includes a fourth sequencing adapter, a fourth sample tag sequence for identifying different samples, and a fourth sequencing primer from its 5'-end to 3'-end. The third downstream primer sequentially includes a third sequencing adapter, a third sample tag sequence for identifying different samples, a third sequencing primer, and a third complementary sequence complementary to the 3'-end of the sequences of the multiple pathogens from its 5'-end to 3'-end. In the third aspect of the present disclosure, the primer set can be used for one-step library construction. Thus, a primer set for detecting multiple pathogens that can reduce aerosol contamination, simplify the library construction steps, and has high accuracy can be provided.

[0017] According to the present disclosure, an amplicon library construction kit, a library construction method, and a primer set for detecting multiple pathogens that can reduce aerosol contamination, simplify the library construction steps, and have high accuracy can be provided. Brief Description of the Drawings

[0018] The present disclosure will now be further explained in detail only by way of examples with reference to the accompanying drawings.

[0019] Figure 1 It is a schematic diagram showing combination form one of the primer combinations involved in the examples of the present disclosure.

[0020] Figure 2 It is a schematic diagram showing combination form two of the primer combinations involved in the examples of the present disclosure.

[0021] Description of the Reference Numerals:

[0022] F…The first upstream primer, 101…The first sequencing adapter, 102…The first sample tag sequence, 103…The first sequencing primer, 104…The first complementary sequence, R1…The first downstream primer, 201…The second sequencing primer, 202…The second complementary sequence, R2…The second downstream primer, 301…The second sequencing adapter, 302…The second sample tag sequence, 201…The second sequencing primer,

[0023] F2…The third upstream primer, 401…The fourth sequencing primer, 402…The fourth complementary sequence, F1…The fourth upstream primer, 501…The fourth sequencing adapter, 502…The fourth sample tag sequence, 401…The fourth sequencing primer, R…The third downstream primer, 601…The third sequencing adapter, 602…The third sample tag sequence, 603…The third sequencing primer, 604…The third complementary sequence. Detailed implementation manners

[0024] The following details the implementation manners of the present disclosure. Examples of the implementation manners are shown in the drawings, where the same or similar reference numerals represent the same or similar elements or elements with the same or similar functions throughout. The implementation manners described through the drawings are exemplary and are only used to explain the present disclosure and should not be construed as a limitation to the present disclosure.

[0025] Those skilled in the art can understand that unless otherwise defined, all terms (including technical terms and scientific terms) used herein have the same meaning as the general understanding of those of ordinary skill in the art in the field to which the present disclosure belongs.

[0026] It should also be understood that terms such as those defined in a general dictionary should be understood to have a meaning consistent with the meaning in the context of the prior art and will not be interpreted with an idealized or overly formal meaning unless defined as here.

[0027] To facilitate the understanding of the present disclosure, the following further explains the present disclosure with specific examples in conjunction with the drawings, and the specific examples do not constitute a limitation to the embodiments of the present disclosure. Those skilled in the art should understand that the drawings are only schematic diagrams of the embodiments, and the components in the drawings are not necessarily essential for implementing the present disclosure.

[0028] The first aspect of the present disclosure provides an amplicon library construction kit. The amplicon library construction kit (which can be abbreviated as "kit") can be used to detect multiple pathogens and / or multiple target genes. The amplicon library construction kit involved in the present disclosure can perform PCR amplification on the target gene sequences of multiple pathogens, so as to facilitate the sequencing of the target gene sequences. Therefore, the amplicon library construction kit of the present disclosure can be called an amplicon library construction kit for detecting multiple pathogens, an amplicon library construction kit for detecting multiple target genes, or an amplicon library construction kit for detecting multiple pathogens / target genes.

[0029] The amplicon library construction kit of the present disclosure has the following technical key points:

[0030] 1. A method for constructing a super-multiplex amplicon library that can achieve co-detection of target genes / pathogenic microorganisms DNA + RNA by one-step amplification. Using the target gene / pathogenic microorganism DNA and RNA in the test sample as templates, the target region and library are amplified using a one-step amplification primer combination or a kit composed of primers. After purifying the amplification products, the amplicon library of the target gene / pathogenic microorganism to be detected is obtained;

[0031] 2. Effectively reduce contamination during library construction and reduce false positive detections. The sample tag sequence (barcode) for differentiating different samples is added at the beginning of PCR in one-step amplification, which can reduce cross-contamination that may occur during library construction and reduce false positive detections;

[0032] 3. High amplification efficiency and high gene detection sensitivity. The three-primer method of the present invention reduces the number of primers, reduces the amplification competition between primers, and improves the amplification efficiency; and the molar ratio of the three primers is 2:1:2 to 5:1:5, preferably 3:1:3. Under this condition, the sample amplification efficiency is the highest. According to experimental verification, a comparative analysis was carried out on the primer concentration ratios of "1:1:1", "2:1:2", "3:1:3", "5:1:5" and "10:1:10". When using this kit to detect samples, the sample amplification efficiency is the highest under the condition of 3:1:3. The detection of 500 copies / mL pathogen is greater than 700 reads (total reads normalized to 1M), or the detection of 10000 copies / mL pathogen is greater than 15000 reads (total reads normalized to 1M).

[0033] 4. Reduce the library construction cost. The one-step method simplifies the steps, reduces the purification and amplification steps, and reduces the usage of PCR instruments, amplification reagents, magnetic beads and various consumables. Compared with the four-primer library construction, the number of three primers is reduced, and the synthesis cost is also lower. One-round amplification reduces the requirements for experimental partitions and reduces the investment in hardware facilities for NGS detection. The overall cost of library construction has greater advantages.

[0034] 5. Can achieve dual-index sequencing. By adding sample tag sequences at both ends of the amplicon, dual-index sequencing can be achieved, which is more widely applicable.

[0035] 6. Save laboratory space. The one-step method reduces the experimental steps and only requires one-round PCR reaction. One laboratory partition can be reduced, reducing the consumption of laboratory space.

[0036] 7 Strong platform compatibility. The kit and primer set of the present invention are applicable to various second-generation sequencing platforms including Ion Torrent, Illumina, and BGI / MGI. The sequencing adapter sequences and universal sequences can be designed according to the differences of each platform.

[0037] 8. The kit is easy to operate and the library construction time is shortened. By combining the two-step amplification into one step, the total number of amplification cycles is reduced, the library construction operation is simplified, the library construction time is shortened to within 4 hours, the rapid sequencing is 5.5 hours, and the rapid analysis is 0.5 hours, and a TAT (Turnaround Time, reporting cycle) time of 10 hours can be achieved.

[0038] The second aspect of the present disclosure provides a method for constructing an amplicon library for detecting multiple pathogens (which can be simply referred to as a library construction method). The library construction method involved in the present disclosure is to enrich and / or add adapters to the target nucleic acid sequence so as to facilitate the sequencing of the target sequence.

[0039] The third aspect of the present disclosure provides a primer set for constructing an amplicon library for detecting multiple pathogens.

[0040] Hereinafter, in conjunction with the drawings, the amplicon library construction kit involved in the present disclosure will be described in detail.

[0041] As described above, the amplicon library construction kit involved in the present disclosure can perform PCR amplification on the target gene sequences of multiple pathogens. In some examples, the target gene sequence can be a gene or gene fragment unique to a certain pathogen, and by detecting the gene or gene fragment, the pathogen can be identified. In some examples, the target gene sequence can include the target gene sequences of multiple pathogens.

[0042] In some examples, the PCR amplification can be multiplex PCR amplification. Multiplex PCR amplification is a technique for simultaneously amplifying multiple target sequences in a single PCR reaction. Multiple pairs of primers can be used in the PCR reaction, with each pair of primers corresponding to a target sequence, to simultaneously amplify multiple different target sequences. Thereby, the detection efficiency can be improved.

[0043] In some examples, the PCR amplification can be ultra-multiplex PCR amplification. Ultra-multiplex PCR amplification is a further technique than multiplex PCR amplification. It can simultaneously amplify more target sequences in a single PCR reaction. Ultra-multiplex PCR amplification usually uses more primer pairs and can amplify dozens or even hundreds or thousands of different target sequences in a single PCR reaction.

[0044] In some examples, the sample types detected by the amplicon library construction kit can be blood, cells, throat swabs, sputum, bronchoalveolar lavage fluid, urine, feces, cerebrospinal fluid, or pleural effusion, etc.

[0045] In some examples, a nucleic acid extraction kit can be used to extract a nucleic acid sample to be tested from a sample. Among them, depending on different sample types, different nucleic acid extraction kits can be used for extraction, or a DNA / RNA co-extraction kit can be used for extraction. In some examples, for a sample containing cells with difficult-to-break cell walls, ultrasonic cell wall breaking can be carried out in advance, and then nucleic acid extraction can be carried out.

[0046] In some examples, the nucleic acid sample to be tested can be stored in a test tube and sealed. In some examples, the nucleic acid sample to be tested can be stored refrigerated or frozen. For example, it can be stored under the conditions of -20°C to -80°C.

[0047] In some examples, the nucleic acid sample to be tested can include at least one of a DNA sample, a cDNA sample, and an RNA sample. In some examples, if the nucleic acid sample to be tested includes an RNA sample, it further includes the step of reverse transcribing the nucleic acid sample to be tested. In some examples, random primers can be used to reverse transcribe the RNA sample. Thus, the nucleic acid sample to be tested containing an RNA sample can be detected.

[0048] In some examples, a fluorescence quantitative kit and a fluorescence quantitative instrument can be used to determine the concentration of nucleic acid in the nucleic acid sample to be tested, and the nucleic acid concentrations of each nucleic acid sample can be made as uniform as possible. The Qubit kit can be used as the fluorescence quantitative kit, and the Qubit instrument can be used as the fluorescence quantitative instrument.

[0049] In some examples, the amplicon library construction kit can include a primer set. In some examples, the primer set can have two combination forms, namely combination form one (which can also be called the first primer set) and combination form two (which can also be called the second primer set). Both combination forms can effectively and efficiently amplify the target fragment.

[0050] Figure 1 is a schematic diagram showing combination form one of the primer involved in the example of the present disclosure.

[0051] In some examples, the first primer set can include a first upstream primer F, a first downstream primer R1, and a second downstream primer R2 (see Figure 1 ). In other words, the primer set can include a first upstream primer F, a first downstream primer R1, and a second downstream primer R2.

[0052] In some examples, the first upstream primer F sequentially includes, from its 5'-end to 3'-end, a first sequencing adapter 101, a first sample tag sequence 102 for identifying different samples, a first sequencing primer 103, and a first complementary sequence 104. In some examples, the first complementary sequence 104 can be a sequence that is complementary to the 5'-end of the target sequence. That is to say, the first complementary sequence 104 can be a sequence that is complementary to the 5'-ends of the sequences of multiple pathogens. Thus, the first upstream primer F can be complementary to the 5'-end of the target sequence.

[0053] In some examples, the first sequencing primer 103 can be a corresponding universal sequence selected according to different sequencing platforms. In some examples, the first sequencing adapter 101 can be a corresponding sequencing adapter selected according to different sequencing platforms. For example, in some examples, the first sequencing adapter 101 and the first sequencing primer 103 can be the sequencing adapter and sequencing primer of the Illumina sequencing platform, the MGI sequencing platform, or the Ion Torrent sequencing platform.

[0054] In some examples, the first downstream primer R1 sequentially includes, from its 5'-end to 3'-end, a second sequencing primer 201 and a second complementary sequence 202. In some examples, the second complementary sequence 202 can be a sequence that is complementary to the 3'-end of the target sequence. That is to say, the second complementary sequence 202 can be a sequence that is complementary to the 3'-ends of the sequences of multiple pathogens. Thus, the first downstream primer R1 can be complementary to the 3'-end of the target sequence. In some examples, the second sequencing primer 201 can be a corresponding universal sequence selected according to different sequencing platforms.

[0055] In some examples, the second downstream primer R2 sequentially includes, from its 5'-end to 3'-end, a second sequencing adapter 301, a second sample tag sequence 302 for identifying different samples, and a second sequencing primer 201. In some examples, the second sequencing adapter 301 can be a corresponding sequencing adapter selected according to different sequencing platforms. For example, in some examples, the second sequencing adapter 301 and the second sequencing primer 201 can be the sequencing adapter and sequencing primer of the Illumina sequencing platform, the MGI sequencing platform, or the Ion Torrent sequencing platform.

[0056] In the first combination form, when amplifying the template DNA / cDNA, the first upstream primer F, the first downstream primer R1, and the second downstream primer R2 in the amplification system are added in a certain proportion. The first upstream primer F and the first downstream primer R1 can amplify the target fragment. After the first upstream primer F binds to the template, it can add the first sample tag sequence 102 and the first sequencing adapter 101 to the target sequence. The first downstream primer R1 and the second downstream primer R2 share the second sequencing primer 201. Therefore, the second downstream primer R2 can use the first downstream primer R1 as a template to add the second sample tag sequence 302 and the second sequencing adapter 301 to the target sequence. Through multiple rounds of PCR amplification, the library construction is finally completed. Refer to Figure 1 , when amplifying the target sequence 100, add the first upstream primer F, the first downstream primer R1, and the second downstream primer R2, and perform one round of PCR amplification to obtain the target library 200.

[0057] In some examples, the molar ratio of the first upstream primer F, the first downstream primer R1, and the second downstream primer R2 can be 2:1:2 to 5:1:5. For example, it can be 2:1:2, 3:1:3, 4:1:4, or 5:1:5. Thus, it is beneficial to improve the amplification efficiency. In some examples, preferably, the molar ratio of the first upstream primer F, the first downstream primer R1, and the second downstream primer R2 can be 3:1:3.

[0058] Figure 2 is a schematic diagram showing the second primer combination form involved in the examples of the present disclosure.

[0059] In some examples, the second primer group may include a fourth upstream primer F1, a third upstream primer F2, and a third downstream primer R (refer to Figure 2 ). In other words, the primer group may include the third upstream primer F2, the fourth upstream primer F1, and the third downstream primer R. It should be noted that, for the convenience of distinguishing between the first combination form and the second combination form, each primer is named with a different numerical number, and the second primer group also includes three primers. In some examples, the second primer group may be composed of the third upstream primer F2, the fourth upstream primer F1, and the third downstream primer R.

[0060] In some examples, the third downstream primer R sequentially includes a third sequencing adapter 601, a third sample tag sequence 602 for identifying different samples, a third sequencing primer 603, and a third complementary sequence 604 from its 5' end to its 3' end. In some examples, the third downstream primer R may be composed of the third sequencing adapter 601, the third sample tag sequence 602, the third sequencing primer 603, and the third complementary sequence 604.

[0061] In some examples, the third complementary sequence 604 can be a sequence that is complementary to the 3'-end of the target sequence. That is to say, the third complementary sequence 604 can be a sequence that is complementary to the 3'-ends of sequences of multiple pathogens. Thus, the third downstream primer R can be complementary to the 3'-end of the target sequence. In some examples, the third sequencing primer 603 can be a corresponding universal sequence selected according to different sequencing platforms. In some examples, the third sequencing adapter 601 can be a corresponding sequencing adapter selected according to different sequencing platforms. For example, in some examples, the third sequencing adapter 601 and the third sequencing primer 603 can be the sequencing adapter and sequencing primer of the Illumina sequencing platform, the MGI sequencing platform, or the Ion Torrent sequencing platform.

[0062] In some examples, the third upstream primer F2 sequentially includes a fourth sequencing primer 401 and a fourth complementary sequence 402 from its 5'-end to its 3'-end. In some examples, the fourth complementary sequence 402 can be a sequence that is complementary to the 5'-end of the target sequence. That is to say, the fourth complementary sequence 402 can be a sequence that is complementary to the 5'-ends of sequences of multiple pathogens. Thus, the third downstream primer R can be complementary to the 5'-end of the target sequence. In some examples, the fourth sequencing primer 401 can be a corresponding universal sequence selected according to different sequencing platforms.

[0063] In some examples, the fourth upstream primer F1 sequentially includes a fourth sequencing adapter 501, a fourth sample tag sequence 502 for identifying different samples, and a fourth sequencing primer 401 from its 5'-end to its 3'-end. In some examples, the fourth sequencing adapter 501 can be a corresponding sequencing adapter selected according to different sequencing platforms. For example, in some examples, the fourth sequencing adapter 501 and the fourth sequencing primer 401 can be the sequencing adapter and sequencing primer of the Illumina sequencing platform, the MGI sequencing platform, or the Ion Torrent sequencing platform. In some examples, the fourth upstream primer F1 can be phosphorylated. Thus, the library can be made adaptable to the MGI sequencing platform.

[0064] In combination form two, when amplifying the template DNA / cDNA, the third upstream primer F2, the fourth upstream primer F1, and the third downstream primer R in the amplification system are added in a certain proportion, and the third upstream primer F2 and the third downstream primer R can amplify the target fragment. The third upstream primer F2 and the fourth upstream primer F1 share the fourth sequencing primer 401, so the fourth upstream primer F1 can use the third upstream primer F2 as a template to add the fourth sample tag sequence 502 and the fourth sequencing adapter 501 to the target sequence; after the third downstream primer R binds to the template, it can add the third sample tag sequence 602 and the third sequencing adapter 601 to the target sequence; through multiple rounds of PCR amplification, the library construction is finally completed. See Figure 2, when amplifying the target sequence 100, add the third upstream primer F2, the fourth upstream primer F1 and the third downstream primer R, and the target library 200 can be obtained by one round of PCR amplification.

[0065] In some examples, the molar ratio of the fourth upstream primer F1, the third upstream primer F2 and the third downstream primer R can be 2:1:2 to 5:1:5. For example, it can be 2:1:2, 3:1:3, 4:1:4, or 5:1:5. Thus, it is beneficial to improve the amplification efficiency. In some examples, preferably, the molar ratio of the fourth upstream primer F1, the third upstream primer F2 and the third downstream primer R can be 3:1:3.

[0066] In some examples, combination form two can be a variant of combination form one. Combination form two and combination form one can be two symmetrically arranged forms. Combination form one includes the first upstream primer F, the first downstream primer R1 and the second downstream primer R2. Correspondingly, combination form two includes the fourth upstream primer F1, the third upstream primer F2 and the third downstream primer R.

[0067] In the present disclosure, the primer sets of combination form one and combination form two can both efficiently construct a library for the target fragment, complete the construction of the amplicon sequencing library through one round of PCR amplification in a reaction tube, and the formed library can perform dual-index sequencing, with a wider adaptability.

[0068] In some examples, the same type of primers designed for different pathogens have similar or the same Tm values. For example, the difference in Tm values between the first upstream primer F for Epstein-Barr virus and the first upstream primer F for Enterovirus is small. In this case, in the PCR reaction, a small range of annealing temperature settings can satisfy the annealing of all amplicons, which is beneficial to improving the amplification efficiency.

[0069] In some examples, multiple gradients of annealing temperature can be set in the PCR reaction. For example, 2 gradients or 3 gradients of annealing temperature can be set. Specifically, when the preset annealing temperature of the primer is 60°C, 2 or 3 temperatures can be selected within the range of 60°C ± 2°C for annealing. In this case, each primer can find the template at the optimal annealing temperature, thereby improving the amplification efficiency.

[0070] In some examples, the amplicon library construction kit may further include a reference sequence. In some examples, the reference sequence may be complementary to the first upstream primer F and the first downstream primer R1 in combination form one. In some examples, the reference sequence may be complementary to the third upstream primer F2 and the third downstream primer R in combination form two. In some examples, the reference sequence may be designed based on the target gene sequences of multiple pathogens, but has a different sequence from the target gene sequences. In this case, the reference sequence can bind to the primer set and can also be distinguished from the target gene sequences. On the one hand, since the primer set is directed against multiple pathogens, when using the primer set for PCR amplification during the experiment, the test sample usually contains only the target gene sequences of one, two (or even zero) pathogens. Therefore, only a small part of the primer sets in the primer set may be able to find a template for binding, and most of the remaining primer sets cannot find a template for binding. At this time, primer dimers may be generated. In the present disclosure, the reference sequence can bind to a part of the primer sets of the primers, playing a role in consuming the primer set, thereby being able to reduce the proportion of primer dimers during the experiment and improve the library quality. On the other hand, the content of the added reference sequence can be a predetermined copy number, that is, the added amount of the reference sequence is known. Subsequently, based on the number of target gene sequences bound to the primer set and the number of reference sequences bound to the primer set, the amount of the corresponding pathogen can be obtained. Thus, quantitative detection of the pathogen can be performed.

[0071] In some examples, both ends of the reference sequence can be respectively bound to the first upstream primer F and the first downstream primer R1 in combination form one, and the reference sequence includes multiple different sequences, having a different sequence within 15 bp from the sequence bound to the first upstream primer F and having a different sequence within 15 bp from the sequence bound to the first downstream primer R1. In this case, when multiple target gene sequences are selected for detection of a pathogen, if, for a pathogen, there is an overlapping region between the multiple selected target gene sequences, at this time, since there are different sequences at both ends of the reference sequence, it is also possible to facilitate the distinction between the sequence of the reference sequence and the target gene sequences, reducing the generation of false positives. In some examples, both ends of the reference sequence can be respectively bound to the third upstream primer F2 and the third downstream primer R in combination form two, and the reference sequence includes multiple different sequences, having a different sequence within 15 bp from the sequence bound to the third upstream primer F2 and having a different sequence within 15 bp from the sequence bound to the third downstream primer R.

[0072] In some examples, the length of the different sequence can be 5 bp. This can facilitate the distinction between the reference sequence and the target gene sequences.

[0073] In some examples, the copy number of each internal reference sequence in the internal reference sequence set is known and can be a predetermined copy number. In some examples, the predetermined copy number can be from 200 copies to 400 copies. Thus, an appropriate amount of the internal reference sequence can be added, which will neither overly affect the binding of the primer to the target gene sequence nor fail to achieve the effect of consuming the primer.

[0074] In some examples, the copy number of the internal reference sequence added to the sample to be tested during library construction is known. Thus, it is convenient to quantify the target gene sequence. Specifically, when adding a primer set and an internal reference sequence set (the added amount is the predetermined copy number) to the sample to be tested for library construction, during the PCR amplification process, ideally, the amplification efficiency of the primer set for a certain target gene sequence and the amplification efficiency of the internal reference sequence corresponding to the target gene sequence are the same; after library construction, the obtained library may contain the sequence of the target gene of a certain pathogen and the internal reference sequence corresponding to the target gene sequence. After sequencing on the machine and analyzing the sequencing data, the number of Reads of the target gene (read segments) and the number of Reads of the internal reference sequence can be obtained; at this time, the copy number of the target gene sequence can be calculated through the relationship of "copy number of target gene sequence / number of Reads of target gene sequence = copy number of internal reference sequence (i.e., the predetermined copy number, which is known) / number of Reads of internal reference sequence", and finally the copy number of the pathogen can be obtained, that is, the content of the pathogen in the sample to be tested can be obtained. Thus, multiple pathogens in the sample can be quantitatively detected. It can be understood that this quantitative detection refers to relative quantification, that is, through the internal reference sequence, the relative magnitudes of the contents of multiple pathogens in a sample can be obtained. In some examples, the copy number of the pathogen can be relatively quantified jointly by the exogenous internal reference Escherichia coli phage MS2 and the amplicon internal reference plasmid.

[0075] In some examples, the amplicon library construction kit may further include at least one of a positive control product, a negative control product, a reverse transcription reagent, a nucleic acid extraction reagent, a library construction reagent (including PCR buffer, DNA polymerase, dNTPs, etc.), a quantification reagent, a purification reagent, and a sequencing reagent. Among them, the positive control product, the negative control product, the reverse transcription reagent, the nucleic acid extraction reagent, the library construction reagent, the quantification reagent, the purification reagent, and the sequencing reagent can be self-made or commercially available.

[0076] In some examples, the positive control product can be a sample including the target gene sequence, and the negative control product can be a sample not including the target gene sequence. Thus, true positive and false positive samples for the target gene sequence can be provided for establishing an ROC curve (Receiver Operating Characteristic curve) to obtain the detection threshold of each target gene sequence, or for conducting positive control experiments or negative control experiments.

[0077] The second aspect of the present disclosure provides a library construction method.

[0078] In some examples, the library construction method may include the following steps: preparing a nucleic acid sample to be tested (which may be abbreviated as the sample to be tested), and obtaining sample DNA (step S100); performing PCR amplification on the sample DNA (step S200); obtaining an amplicon library (step S300). Thus, a target library can be obtained.

[0079] In some examples, in step S100, the sample DNA can be obtained from the object to be tested. For example, the sample DNA can be obtained by collecting samples containing tissues, body fluids, etc. of the object to be tested. The sample types usually may include blood, tissue sections, effusions, throat swabs, sputum, nasal swabs, etc.

[0080] In some examples, a nucleic acid extraction kit can be used to extract and obtain the sample DNA. Among them, different nucleic acid extraction kits can be used for extraction according to different sample types, or a DNA / RNA co-extraction kit can be used for extraction. In some examples, for samples containing cells with difficult-to-break walls, ultrasonic wall-breaking can be carried out in advance, and then nucleic acid extraction can be carried out. In some examples, the nucleic acid sample to be tested obtained by extraction can be stored in a test tube and sealed. In some examples, the nucleic acid sample to be tested can be stored refrigerated or frozen. For example, it can be stored under the condition of -20°C to -80°C.

[0081] In some examples, the sample to be tested may include at least one of a DNA sample and an RNA sample. In some examples, if the nucleic acid sample to be tested includes an RNA sample, it further includes the step of reverse transcribing the nucleic acid sample to be tested. That is to say, when the nucleic acid sample to be tested includes an RNA sample, the RNA sample can be reverse transcribed first. In some examples, random primers can be used to reverse transcribe the RNA sample. Thus, the sample to be tested containing an RNA sample can be detected.

[0082] In some examples, a fluorescence quantitative kit and a fluorescence quantifier can be used to measure the concentration of nucleic acid in the sample to be tested, and the nucleic acid concentrations of each sample to be tested are made as uniform as possible. A Qubit kit can be used as the fluorescence quantitative kit, and a Qubit instrument can be used as the fluorescence quantifier.

[0083] In some examples, in step S200, a primer set can be added for PCR amplification. In the present disclosure, all primers can be added in the same step for one-step amplification to obtain the target library.

[0084] In some examples, in step S200, the primer set can be the same as the primer set in the kit described in the first aspect of the present disclosure. For the composition of the relevant structure and the library construction conditions, reference can be made to the previous description and will not be elaborated here. In some examples, in step S200, the kit of the first aspect of the present disclosure can be used to perform PCR amplification on the sample DNA. In the second aspect of the present disclosure, an amplicon library can be obtained by using a one-step library construction method. Thus, a method for constructing an amplicon library for detecting multiple pathogens can be provided, which can reduce aerosol contamination, simplify the library construction steps, and have high accuracy.

[0085] In some examples, after step S200 and before step S300, a purification step can be performed. Thus, non-specific molecules or impurities (such as primer dimers, dNTPs) can be removed through the purification step, and the target library can be purified, thereby reducing background impurities and improving the accuracy and reliability of subsequent experiments.

[0086] The third aspect of the present disclosure provides a primer set. The primer set can be used for constructing an amplicon library for detecting multiple pathogens. In some examples, the primer set can include multiple primers. The multiple primers can have two combination forms, namely combination form one (which can also be called the first primer set) and combination form two (which can also be called the second primer set). Both combination forms can effectively and efficiently amplify the target fragment. The primer set involved in the third aspect of the present disclosure is the same as the primer set in the kit described in the first aspect of the present disclosure. For the composition of the relevant structure and the library construction conditions, reference can be made to the previous description and will not be elaborated here. In the third aspect of the present disclosure, the primer set can be used for one-step library construction. Thus, a primer set for detecting multiple pathogens can be provided, which can reduce aerosol contamination, simplify the library construction steps, and have high accuracy.

[0087] In summary, the present disclosure can provide an amplicon library construction kit, a library construction method, and a primer set for detecting multiple pathogens, which can reduce aerosol contamination, simplify the library construction steps, and have high accuracy.

[0088] Next, the above-mentioned library construction method and primer set involved in the present disclosure will be further explained in detail in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present disclosure.

[0089] [Example 1] 50-microorganism system

[0090] (1) Primer design

[0091] In this example, primer design is carried out based on the illumina sequencing platform. The combination form one is adopted for this library construction, and the primer structure composition is shown in Table 1.

[0092] Table 1

[0093]

[0094]

[0095] To achieve dual-index sequencing, a first sample tag sequence is added to the first upstream primer F. To ensure base balance in sequencing, at least 4 sets of the first upstream primer F are required in each sequencing library, and the first sample tag sequences of these 4 sets need to ensure that 4 different bases are contained at each identical position.

[0096] In this embodiment, primers are designed for 50 pathogens, with 2 pairs of primers designed for each pathogen. At the same time, 2 pairs of primers are designed for the human sex gene SRY as sex verification primers, 1 pair of primers is designed for each of the human conserved genes β-Actin and GAPDH as reverse transcription verification primers, and 1 pair of primers is designed for each of the coat gene and proA gene of the Escherichia coli phage MS2 as exogenous internal reference primers, for a total of 106 amplicon primer pairs. Information on the specific primers is shown in the following table (only one set of upstream primer groups is shown). The first complementary sequence of the first upstream primer F is shown in Table 2.

[0097] Table 2

[0098]

[0099]

[0100]

[0101] The second complementary sequence of the first downstream primer R1 is shown in Table 3.

[0102] Table 3

[0103]

[0104]

[0105]

[0106] (2) Sample Selection

[0107] Select 20 pathogens, including Staphylococcus aureus, Mycobacterium tuberculosis complex, Streptococcus pneumoniae, Haemophilus influenzae, Pseudomonas aeruginosa, Klebsiella pneumoniae, Streptococcus pyogenes, Epstein-Barr virus, Influenza A virus H1N1, Influenza B virus, Human herpes simplex virus 1, Human herpes simplex virus 2, Human respiratory syncytial virus A, Human respiratory syncytial virus B, Human coronavirus 229E, Human coronavirus NL63, Human coronavirus OC43, Coronavirus HKU1, Mycoplasma pneumoniae, Aspergillus flavus (source: Shenzhen Center for Disease Control and Prevention, Beijing Nano Biological Technology Co., Ltd., Guangzhou Bondeson Biotech Co., Ltd. and National Institute of Metrology, China). Another 30 pathogens (Enterovirus, Human parvovirus B19, WU polyomavirus, Bordetella holmesii, Influenza C virus, Rhinovirus A, Rhinovirus B, Rhinovirus C, Bordetella pertussis, Human parechovirus A, Chlamydia psittaci, Corynebacterium striatum, Serratia marcescens, Mycobacterium scrofulaceum, Klebsiella aerogenes, Pasteurella multocida, Chlamydia trachomatis, Orientia tsutsugamushi, Burkholderia pseudomallei, Streptococcus agalactiae, Candida auris, Aspergillus fumigatus, Middle East respiratory syndrome coronavirus, Enterovirus 71, Mycobacterium abscessus, Chlamydia pneumoniae, Mumps virus, Bordetella parapertussis, Bordetella bronchiseptica, Neisseria meningitidis), synthetic plasmids. Prepare a medium-concentration (10,000 ± 2,000 copies / mL) mixed sample from the above 50 pathogens. Add the same amount of exogenous internal reference to the sample, first perform reverse transcription, and then use a one-step method to construct a library. Set different primer ratios in the PCR system, namely primer concentration ratios of "1:1:1", "2:1:2", "3:1:3", "5:1:5", and "10:1:10" for comparative analysis.

[0108] (3) Construct a library

[0109] Prepare the PCR reaction system as shown in the following table:

[0110]

[0111] Use the PCR reaction solution to construct a library for the medium-concentration (10,000 ± 2,000 copies / mL) mixed sample. The PCR reaction program is as shown in the following table:

[0112]

[0113]

[0114] Take 25 μl of the PCR product, supplement TE to 50 μl, purify it using magnetic beads, rinse it with 80% ethanol, and elute it with TE after the magnetic beads are dried to obtain the target library (amplicon library).

[0115] (4) Quantification, fragment analysis, and sequencing

[0116] Refer to the Qubit Flurometer 3.0 instruction manual to measure the concentration of the purified product.

[0117] According to the quantification results, the amplicon libraries of all samples were uniformly diluted to the same molar concentration, and the same volume of each sample library was mixed in a 1:1 ratio. Next, the library was denatured according to the requirements for loading. The diluted and denatured library was sequenced on the Illumina sequencing platform.

[0118] The results of all libraries after sequencing were compared to analyze the proportion of invalid data and amplification efficiency at different primer concentration ratios. The total number of reads in the sequencing results of each sample was normalized to one million (normalized reads per million, nRPM). The results are shown in Tables 4 and 5.

[0119] Table 4

[0120]

[0121] Table 5

[0122]

[0123]

[0124]

[0125]

[0126]

[0127]

[0128]

[0129]

[0130]

[0131]

[0132]

[0133]

[0134]

[0135]

[0136] The results showed that for primer concentration ratios of "2:1:2", "3:1:3", and "5:1:5", the proportion of valid data in the one-step library construction was over 80%, and all corresponding microorganisms could be detected, and the relative concentration results were consistent with the sample concentration. Among them, for the primer concentration ratio of "3:1:3", the proportion of valid data was the highest, the nRPM was the highest, and the amplification effect was the best.

[0137] [Example 2] 40-Microorganism System

[0138] (1) Primer Design

[0139] a. One-step library construction:

[0140] Select the primer sets for detecting 40 common microorganisms in bloodstream infections to build and test the system for one-step library construction reactions.

[0141] In this example, primer design was carried out based on the illumina sequencing platform. The library construction in this article adopted combination form one, and the specific primer structure composition can be seen in Table 1 of Example 1.

[0142] In this example, primers were designed for 40 pathogens, 2 pairs of primers were designed for each pathogen, and at the same time, 2 pairs of primers were designed for the human sex gene SRY as sex verification primers, 1 pair of primers were designed for the human conserved genes β-Actin and GAPDH respectively as reverse transcription verification primers, and 1 pair of primers were designed for the coat gene and proA gene of Escherichia coli phage MS2 respectively as exogenous internal reference primers, for a total of 86 amplicon primer pairs.

[0143] The information on the specific primers is shown in the following table (only one set of upstream primer sets is shown). The first complementary sequence of the first upstream primer F and the second complementary sequence of the first downstream primer R1 are shown in Table 6-1 and Table 6-2.

[0144] Table 6-1

[0145]

[0146]

[0147]

[0148] Table 6-2

[0149]

[0150]

[0151]

[0152] b. Control group: Two-step library construction

[0153] In the control group, five primers were used for two-step PCR amplification, namely the first primer, the second primer, the third primer, the fourth primer, and the fifth primer. The first primer, the second primer, and the third primer were used in the first round of PCR amplification, and the fourth primer and the fifth primer were used in the second round of PCR amplification.

[0154] The primer structures involved are as follows:

[0155] The first primer consists of a universal sequence 1 (i.e., the first sequencing primer) and an upstream specific primer sequence (i.e., the first complementary sequence) in sequence from the 5' end to the 3' end; the second primer consists of a universal sequence 2 (i.e., the second sequencing primer) and a downstream specific primer sequence (i.e., the second complementary sequence) in sequence from the 5' end to the 3' end; the third primer consists of a sequencing adapter sequence 2 (i.e., the second sequencing adapter), a sample tag sequence 1 (i.e., the second sample tag sequence), and a universal sequence 2 in sequence from the 5' end to the 3' end; the fourth primer consists of a sequencing adapter sequence 1 (i.e., the first sequencing adapter), a sample tag sequence 2 (i.e., the first sample tag sequence), and a universal sequence 1 in sequence from the 5' end to the 3' end; the fifth primer is a sequencing adapter sequence 2. Among them, the first primer and the second primer need to be designed and synthesized according to the non-specific sequence of the target amplicon, and the first complementary sequence and the second complementary sequence therein are the same as the first complementary sequence and the second complementary sequence in Table 6 of the one-step library construction. The third primer, the fourth primer, and the fifth primer are universal primers that can be used for each sample, and the specific sequence information is also the same as that of the one-step library construction.

[0156] (2) Sample-related

[0157] Twelve pathogens were selected, including Staphylococcus aureus, Mycobacterium tuberculosis complex, Streptococcus pneumoniae, Haemophilus influenzae, Pseudomonas aeruginosa, Escherichia coli, Enterococcus faecalis, Klebsiella pneumoniae, Streptococcus pyogenes, Aspergillus flavus, Epstein-Barr virus, and Human herpes simplex virus 1 (source: Shenzhen Center for Disease Control and Prevention, Guangzhou Bondsen Biotechnology Co., Ltd., and Beijing Na Biotechnology Co., Ltd.). Using negative whole blood samples as the dilution matrix, mixed samples with two concentration levels were prepared, namely medium concentration (10000 ± 2000 copies / mL) and low concentration (500 ± 100 copies / mL). The same amount of exogenous internal reference was added to the two concentration mixed samples. After nucleic acid extraction and reverse transcription, the one-step library construction and the traditional two-step PCR method were used respectively.

[0158] Among the 40 pathogens, another 28 pathogens (Klebsiella oxytoca, Klebsiella aerogenes, coagulase-negative staphylococci, Acinetobacter baumannii, Enterococcus spp., Enterococcus faecium, Enterobacter cloacae, Stenotrophomonas maltophilia, Streptococcus agalactiae, viridans streptococci group, Serratia marcescens, Burkholderia cepacia complex, Listeria monocytogenes, Proteus mirabilis, Bacteroides vulgatus, Bacteroides fragilis, Salmonella enterica, Prevotella spp., Citrobacter spp., Bacillus cereus group, Cryptococcus neoformans, Candida spp., Pneumocystis jirovecii, Mucor spp., Rhizopus spp., Rhizomucor spp., Lichtheimia spp., cytomegalovirus) were all artificially synthesized plasmids. Using negative whole blood samples as the dilution matrix, medium-concentration (10,000 ± 2,000 copies / mL) simulated mixed samples were prepared. The same amount of exogenous internal reference was added to the simulated samples. After nucleic acid extraction and reverse transcription, a one-step library construction method was used.

[0159] (3) Library construction

[0160] a. PCR program for one-step library construction:

[0161] Prepare the PCR reaction system as shown in the following table:

[0162] Component Volume PCR Mix 6uL First upstream primer F Mix (50μM) 3μL First downstream primer R1Mix (50μM) 1μL Second downstream primer R2 Mix (50μM) 3μL Reverse transcription product 15μL <![CDATA[DNAase-free H2O]]> 2μL Total 30μL

[0163] Use the PCR reaction solution for one-step amplification to construct libraries for mixed samples (medium concentration (10,000 ± 2,000 copies / mL) and low concentration (500 ± 100 copies / mL)) and mixed simulated samples of simulated mixed samples. The PCR reaction program is the same as that in Example 1.

[0164] Take 25 μl of the PCR product, supplement TE to 50 μl, purify using magnetic beads, rinse with 80% ethanol, and elute with TE after the magnetic beads are dried to obtain the target library (amplicon library).

[0165] b. PCR program for two-step library construction:

[0166] Prepare the first-round PCR reaction system as shown in the following table:

[0167]

[0168]

[0169] Place the PCR tube in the PCR instrument, set the following program and run.

[0170]

[0171] Purify the first-round PCR product twice. The purification steps are the same as those in the one-step method, and 18 μL of the product is eluted.

[0172] The reaction system for the second round of PCR is shown in the following table:

[0173] Component Volume PCR Mix 12.5uL Fourth primer F2 Mix (10μM) 2.5μL Fifth primer R3 Mix (10μM) 2.5μL First-round PCR purified product 7.5μL Total 25μL

[0174] Place the PCR tube in the PCR instrument, set the following program and run it.

[0175]

[0176] Purify and quantify the amplicon library obtained from the second round of PCR. The method is the same as the purification operation in the one-step method to obtain the target library of the two-step method.

[0177] Compared with the "two rounds of PCR and three rounds of purification" of the two-step method, the one-step method with "one round of PCR and two rounds of purification" saves a lot of time for manual operations, reducing the library construction time from 6 hours in the two-step method to 4 hours.

[0178] (4) Quantification, fragment analysis and sequencing

[0179] Refer to the Qubit Flurometer 3.0 instruction manual to measure the concentration of the purified product.

[0180] According to the quantification results of the quantified library, uniformly dilute the amplicon libraries of all samples to the same molar concentration, take the same volume of each sample library and mix them in a 1:1 ratio. Next, denature the library according to the requirements for loading. Sequence the diluted and denatured library using the Illumina sequencing platform.

[0181] Compare the results of sequencing all libraries of the medium-concentration and low-concentration mixed samples to analyze the feasibility of the one-step method. The total number of reads in the sequencing results of each sample is normalized to one million (normalized reads per million, nRPM). The results are shown in Table 7.

[0182] Analyze the results of the one-step library construction and sequencing of the simulated mixed sample library. The results are shown in Table 8.

[0183] Table 7

[0184]

[0185]

[0186]

[0187]

[0188]

[0189] As shown in Table 7, the results show that the detection results of the one-step method are consistent with those of the two-step method. Both methods can detect the corresponding microorganisms, and the relative concentration results are consistent with the sample concentration. The consistency of the two detection methods is 100%.

[0190] Table 8

[0191]

[0192]

[0193]

[0194]

[0195]

[0196] As shown in Table 8, the results show that the one-step method can detect the corresponding microorganisms, and the relative concentration results are consistent with the sample concentration, meeting the expectations.

[0197] [Example 3] 127 Pathogens and 8 Drug Resistance Gene Systems

[0198] Select the detection primer sets for 127 common pathogens and 8 drug resistance genes in respiratory tract infections to build and test a one-step library construction reaction system.

[0199] (1) Primer Design

[0200] In this example, primer design is carried out based on the BGI MGI sequencing platform. The primer set in Form II is used for library construction, and the primer structure composition is shown in Table 9.

[0201] Table 9

[0202]

[0203] To achieve dual-index sequencing, a third sample tag sequence is added to the third downstream primer R. To ensure the base balance of sequencing, at least 4 sets of the third downstream primer R are required in each sequenced library, and the third sample tag sequences of these 4 sets need to ensure that each same position contains 4 different bases.

[0204] In this example, primers are designed for 127 pathogens and 8 drug resistance genes. 2 pairs of primers are designed for each pathogen and drug resistance gene. At the same time, 2 pairs of primers are designed for the human sex gene SRY as sex verification primers, 1 pair of primers are designed for the human conserved genes β-Actin and GAPDH respectively as reverse transcription verification primers, and 1 pair of primers are designed for the coat gene and proA gene of Escherichia coli phage MS2 respectively as exogenous internal reference primers, totaling 276 amplicon primer pairs.

[0205] The fourth complementary sequence of the third upstream primer F2 and the third complementary sequence of the third downstream primer R (only one set of downstream primer groups is shown) are shown in Tables 10-1 and 10-2.

[0206] Table 10-1

[0207]

[0208]

[0209]

[0210]

[0211]

[0212]

[0213]

[0214] Table 10-2

[0215]

[0216]

[0217]

[0218]

[0219]

[0220]

[0221]

[0222] (2) Sample-related

[0223] Twenty pathogens were selected, including Staphylococcus aureus, Mycobacterium tuberculosis complex, Streptococcus pneumoniae, Haemophilus influenzae, Pseudomonas aeruginosa, Klebsiella pneumoniae, Streptococcus pyogenes, Epstein-Barr virus, Influenza A virus H1N1, Influenza B virus, Human herpes simplex virus 1, Human herpes simplex virus 2, Human respiratory syncytial virus A, Human respiratory syncytial virus B, Human coronavirus 229E, Human coronavirus NL63, Human coronavirus OC43, Coronavirus HKU1, Mycoplasma pneumoniae, Aspergillus flavus (from the same source as in Example 1). Using negative alveolar lavage fluid as the dilution matrix, mixed samples at two concentration levels were prepared, namely medium concentration (10000±2000 copies / mL) and low concentration (500±100 copies / mL). The same amount of exogenous internal reference was added to the two concentration mixed samples. After nucleic acid extraction and reverse transcription, one-step library construction and two-step library construction were carried out respectively. The one-step amplification system and PCR program are as described in Example 1.

[0224] Among the 127 pathogens and 8 drug-resistant genes, another 107 pathogens and 8 drug-resistant genes (varicella-zoster virus, cytomegalovirus, Enterovirus, Enterovirus D68, Human herpesvirus 6, Escherichia coli, Moraxella catarrhalis, Enterovirus A, Enterovirus B, Enterovirus C, Human parvovirus B19, WU polyomavirus, Bordetella holmesii, Influenza C virus, Rhinovirus A, Rhinovirus B, Rhinovirus C, Histoplasma capsulatum, Mucor racemosus, Rhizopus oryzae, Bordetella pertussis, Talaromyces marneffei, Human parechovirus A, Human herpesvirus 7, BK polyomavirus, Enterobacter cloacae, Chlamydia psittaci, Corynebacterium striatum, Serratia marcescens, Mycobacterium scrofulaceum, Mycobacterium fortuitum, Klebsiella aerogenes, Mycobacterium smegmatis, Klebsiella oxytoca, Scedosporium apiospermum, Pasteurella multocida, Tropheryma whipplei, Elizabethkingia meningoseptica, Chlamydia trachomatis, Orientia tsutsugamushi, Burkholderia mallei, Proteus mirabilis, Burkholderia pseudomallei, JC polyomavirus, Rhodococcus equi, Rickettsia rickettsii, Rickettsia prowazekii, Nocardia gelsenkirchenensis, Nocardia asteroides, Nocardia fossor, Nocardia brasiliensis, Nocardia farcinica, Cryptococcus gattii complex, Enterococcus faecalis, Rickettsia typhi, Enterococcus faecium, Staphylococcus epidermidis, Staphylococcus haemolyticus, Enterovirus D, Burkholderia cepacia complex, Stenotrophomonas maltophilia, Aspergillus, Streptococcus agalactiae, Salmonella enterica subsp. enterica, Candida albicans, Candida glabrata, Candida auris, Aspergillus fumigatus, KPC, NDM, vanA, vanB, qnrS, Acinetobacter baumannii, mecA, mcr, macrolides, Middle East respiratory syndrome coronavirus, SARS coronavirus, Coronavirus disease 2019, Omicron variant of SARS-CoV-2, Enterovirus 71, Influenza A virus, Coxiella burnetii, Francisella tularensis, Human parainfluenza virus 1, Human parainfluenza virus 2, Human parainfluenza virus 3, Human parainfluenza virus 4, Human metapneumovirus, Bocavirus, Rubella virus, Measles virus, Mycobacterium abscessus, Mycobacterium kansasii, Cryptococcus neoformans, Pneumocystis jirovecii, Chlamydophila pneumoniae, Human adenovirus B, Human adenovirus C, Human adenovirus E, Mumps virus, Bordetella parapertussis, Bordetella bronchiseptica, Legionella pneumophila, Nocardia, Mycobacterium bufis, Mycobacterium intracellulare, Mycobacterium avium, Mycobacterium gordonae, Mycobacterium chelonae, Neisseria meningitidis, Influenza A virus H3N2, Influenza A virus H5N1 and Influenza A virus H7N9), synthetic plasmids, using negative alveolar lavage fluid as a dilution matrix, prepare a medium-concentration (10,000 ± 2,000 copies / mL) simulated mixed sample. The same amount of exogenous internal reference is added to the simulated sample. After nucleic acid extraction and reverse transcription, a one-step library construction method is used.

[0225] (3) Construct a library

[0226] Prepare the PCR reaction system as shown in the following table:

[0227]

[0228] Construct a library from the PCR reaction solution of one-step amplification for mixed samples with low concentration (500 ± 100 copies / mL) and medium concentration (10,000 ± 2,000 copies / mL). The PCR reaction program is the same as that in Example 1. And purify the PCR product as in Example 1 to obtain the target library (amplicon library).

[0229] Establish a control group for two-step library construction. The primer design and library construction method are the same as those of the control group for two-step library construction in Example 2, only replacing the upstream and downstream complementary sequences with the upstream and downstream complementary sequences shown in Table 9.

[0230] (4) Quantification, fragment analysis, and sequencing

[0231] Refer to the Qubit Flurometer 3.0 manual to measure the concentration of the purified product.

[0232] According to the quantification results of the quantified library, uniformly dilute the amplicon libraries of all samples to the same molar concentration, take the same volume of each sample library and mix them in a 1:1 ratio. Next, denature the library according to the requirements for loading. Sequence the diluted and denatured library using the Illumina sequencing platform.

[0233] Compare the results of all sequenced libraries to analyze the proportion of invalid data and amplification efficiency at different primer concentration ratios. The total number of reads in the sequencing results of each sample is normalized to one million (normalized reads per million, nRPM). The results are shown in Table 11.

[0234] Analyze the results of the one-step library construction and sequencing of the simulated mixed sample library. The results are shown in Table 12.

[0235] Table 11

[0236]

[0237]

[0238]

[0239]

[0240]

[0241]

[0242]

[0243]

[0244] As shown in Table 11, the results show that the detection results of the one-step method are consistent with those of the two-step method. Both can detect the corresponding microorganisms, and the relative concentration results are consistent with the sample concentration. The consistency of the two detection methods is 100%.

[0245] Table 12

[0246]

[0247]

[0248]

[0249]

[0250]

[0251]

[0252]

[0253]

[0254]

[0255]

[0256]

[0257]

[0258]

[0259]

[0260] As shown in Table 12, the results show that the one-step method can detect the corresponding microorganisms, and the relative concentration results are consistent with the sample concentration, meeting the expectations.

[0261] In summary, the one-step library construction kit and method provided by the present invention are easy to operate, have a short library construction time, high amplification efficiency, and high gene detection sensitivity, and can be used for the detection of various pathogens.

[0262] Although the present disclosure has been specifically described above in connection with the accompanying drawings and embodiments, it is to be understood that the above description does not limit the present disclosure in any way. Those skilled in the art can make modifications and variations to the present disclosure as needed without departing from the essence and scope of the present disclosure, and these modifications and variations all fall within the scope of the present disclosure.

Claims

1. An amplicon library construction kit for detecting multiple pathogens, characterized in that, The kit includes the first primer set or the second primer set. The first primer set includes a first upstream primer, a first downstream primer, and a second downstream primer. The first upstream primer sequentially includes a first sequencing adapter, a first sample tag sequence for identifying different samples, a first sequencing primer, and a first complementary sequence complementary to the 5'-end of the sequences of the multiple pathogens from its 5'-end to 3'-end. The first downstream primer sequentially includes a second sequencing primer and a second complementary sequence complementary to the 3'-end of the sequences of the multiple pathogens from its 5'-end to 3'-end. The second downstream primer sequentially includes a second sequencing adapter, a second sample tag sequence for identifying different samples, and a second sequencing primer from its 5'-end to 3'-end. The second primer set includes a third upstream primer, a fourth upstream primer, and a third downstream primer. The third upstream primer sequentially includes a fourth sequencing primer and a fourth complementary sequence complementary to the 5'-end of the sequences of the multiple pathogens from its 5'-end to 3'-end. The fourth upstream primer sequentially includes a fourth sequencing adapter, a fourth sample tag sequence for identifying different samples, and a fourth sequencing primer from its 5'-end to 3'-end. The third downstream primer sequentially includes a third sequencing adapter, a third sample tag sequence for identifying different samples, a third sequencing primer, and a third complementary sequence complementary to the 3'-end of the sequences of the multiple pathogens from its 5'-end to 3'-end.

2. The amplicon library construction kit according to claim 1, wherein The first sequencing adapter, the first sequencing primer, the second sequencing adapter, the second sequencing primer, the third sequencing adapter, the third sequencing primer, the fourth sequencing adapter, and the fourth sequencing primer are sequencing adapters and sequencing primers of the Illumina sequencing platform, the MGI sequencing platform, or the IonTorrent sequencing platform.

3. The amplicon library construction kit according to claim 1, wherein The amplicon library construction kit further includes an internal reference sequence that can be complementary to the first upstream primer and the first downstream primer, or to the third upstream primer and the third downstream primer, and the internal reference sequence has a different sequence from the gene sequences of the multiple pathogens.

4. The amplicon library construction kit according to claim 1, wherein the multiple pathogens include Epstein-Barr virus, Klebsiella aerogenes, Streptococcus agalactiae, Mycobacterium tuberculosis complex, Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa, Haemophilus influenzae, Streptococcus pyogenes, Streptococcus pneumoniae, Human herpes simplex virus 1, Cytomegalovirus, Escherichia coli, Enterobacter cloacae, Klebsiella oxytoca, Proteus mirabilis, Enterococcus faecalis, Enterococcus faecium, Burkholderia cepacia complex, Stenotrophomonas maltophilia, Aspergillus, Acinetobacter baumannii, Cryptococcus neoformans, Pneumocystis jirovecii, Serratia marcescens, Listeria monocytogenes, Bacillus cereus group, Bacteroides fragilis, Mucor, Rhizopus, Candida, Lichtheimia, Rhizomucor, Coagulase-negative staphylococci, Enterococcus, Viridans streptococci group, Bacteroides vulgatus, Salmonella enterica, Prevotella, Citrobacter.

5. The amplicon library construction kit according to claim 4, wherein In the first primer set, the first complementary sequence is SEQ ID NO.7, SEQ ID NO.15, SEQ ID NO.44, SEQ ID NO.45, SEQ ID NO.54, SEQ ID NO.55, SEQ ID NO.82, SEQ ID NO.83, SEQ ID NO.86 to SEQ ID NO.89, SEQ ID NO.100 to SEQ ID NO.109, SEQ ID NO.225 to SEQ ID NO.282, and the second complementary sequence is SEQ ID NO.9, SEQ ID NO.120, SEQ ID NO.149, SEQ ID NO.150, SEQ ID NO.159, SEQ ID NO.160, SEQ ID NO.187, SEQ ID NO.188, SEQ ID NO.191 to SEQ ID NO.194, SEQ ID NO.205 to SEQ ID NO.214, SEQ ID NO.283 to SEQ ID NO.

340.

6. The amplicon library construction kit according to claim 1, wherein The multiple pathogens include Staphylococcus aureus, Mycobacterium tuberculosis complex, Streptococcus pneumoniae, Haemophilus influenzae, Pseudomonas aeruginosa, Klebsiella pneumoniae, Streptococcus pyogenes, Epstein - Barr virus, Influenza A virus H1N1, Influenza B virus, Human herpesvirus 1, Human herpesvirus 2, Human respiratory syncytial virus A, Human respiratory syncytial virus B, Human coronavirus 229E, Human coronavirus NL63, Human coronavirus OC43, Coronavirus HKU1, Mycoplasma pneumoniae, Aspergillus flavus, Varicella - zoster virus, Cytomegalovirus, Enterovirus genus, Enterovirus D68, Human herpesvirus 6, Escherichia coli, Moraxella catarrhalis, Enterovirus group A, Enterovirus group B, Enterovirus group C, Human parvovirus B19, WU polyomavirus, Bordetella holmesii, Influenza C virus, Rhinovirus A, Rhinovirus B, Rhinovirus C, Histoplasma capsulatum, Mucor racemosus, Rhizopus oryzae, Bordetella pertussis, Talaromyces marneffei, Human parechovirus A, Human herpesvirus 7, BK polyomavirus, Enterobacter cloacae, Chlamydia psittaci, Corynebacterium striatum, Serratia marcescens, Mycobacterium scrofulaceum, Mycobacterium fortuitum, Klebsiella aerogenes, Mycobacterium smegmatis, Klebsiella oxytoca, Scedosporium apiospermum, Pasteurella multocida, Tropheryma whipplei, Elizabethkingia meningoseptica, Chlamydia trachomatis, Orientia tsutsugamushi, Burkholderia mallei, Proteus mirabilis, Burkholderia pseudomallei, JC polyomavirus, Rhodococcus equi, Rickettsia rickettsii, Rickettsia prowazekii, Nocardia gelsenkirchenensis, Nocardia asteroides, Nocardia fossicola, Nocardia brasiliensis, Nocardia farcinica, Cryptococcus gattii complex, Enterococcus faecalis, Rickettsia typhi, Enterococcus faecium, Staphylococcus epidermidis, Staphylococcus haemolyticus, Enterovirus group D, Burkholderia cepacia complex, Stenotrophomonas maltophilia, Aspergillus genus, Streptococcus agalactiae, Salmonella enterica subsp. enterica, Candida albicans, Candida glabrata, Candida auris, Aspergillus fumigatus, KPC, NDM, vanA, vanB, qnrS, Acinetobacter baumannii, mecA, mcr, Macrolides, Middle East respiratory syndrome coronavirus, SARS coronavirus, Coronavirus disease 2019, Omicron variant of SARS - CoV - 2, Enterovirus 71, Influenza A virus, Coxiella burnetii, Francisella tularensis, Human parainfluenza virus 1, Human parainfluenza virus 2, Human parainfluenza virus 3, Human parainfluenza virus 4, Human metapneumovirus, Bocavirus genus, Rubella virus, Measles virus, Mycobacterium abscessus, Mycobacterium kansasii, Cryptococcus neoformans, Pneumocystis jirovecii, Chlamydophila pneumoniae, Human adenovirus group B, Human adenovirus group C, Human adenovirus group E, Mumps virus, Bordetella parapertussis, Bordetella bronchiseptica, Legionella pneumophila, Nocardia genus, Mycobacterium xenopi, Mycobacterium intracellulare, Mycobacterium avium, Mycobacterium gordonae, Mycobacterium chelonae, Neisseria meningitidis, Influenza A virus H3N2, Influenza A virus H5N1, and Influenza A virus H7N9.

7. The amplicon library construction kit according to claim 6, wherein In the second primer set, the third complementary sequence is SEQ ID NO.7, SEQ ID NOs.15 to 113, SEQ ID NOs.225 to 250, SEQ ID NOs.346 to 489, and the fourth complementary sequence is SEQ ID NO.9, SEQ ID NOs.120 to 218, SEQ ID NOs.283 to 308, SEQ ID NOs.490 to 633.

8. The amplicon library construction kit according to claim 1, wherein In the second primer set, the fourth upstream primer is phosphorylated.

9. A method for constructing an amplicon library for detecting multiple pathogens, characterized in that, Comprising the following steps: Prepare a nucleic acid sample to be tested, obtain sample DNA, wherein when the nucleic acid sample to be tested includes an RNA sample, the RNA sample is first reverse-transcribed; use the kit according to any one of claims 1-8 to perform PCR amplification on the sample DNA; obtain an amplicon library.

10. A primer set for constructing an amplicon library for detecting multiple pathogens, characterized in that, The primer set has two combination forms, namely combination form one and combination form two. Combination form one includes a first upstream primer, a first downstream primer, and a second downstream primer. The first upstream primer sequentially includes a first sequencing adapter, a first sample tag sequence for identifying different samples, a first sequencing primer, and a first complementary sequence complementary to the 5'-end of the sequences of the multiple pathogens from its 5'-end to 3'-end. The first downstream primer sequentially includes a second sequencing primer and a second complementary sequence complementary to the 3'-end of the sequences of the multiple pathogens from its 5'-end to 3'-end. The second downstream primer sequentially includes a second sequencing adapter, a second sample tag sequence for identifying different samples, and a second sequencing primer from its 5'-end to 3'-end. Combination form two includes a third upstream primer, a fourth upstream primer, and a third downstream primer. The third upstream primer sequentially includes a fourth sequencing primer and a fourth complementary sequence complementary to the 5'-end of the sequences of the multiple pathogens from its 5'-end to 3'-end. The fourth upstream primer sequentially includes a fourth sequencing adapter, a fourth sample tag sequence for identifying different samples, and a fourth sequencing primer from its 5'-end to 3'-end. The third downstream primer sequentially includes a third sequencing adapter, a third sample tag sequence for identifying different samples, a third sequencing primer, and a third complementary sequence complementary to the 3'-end of the sequences of the multiple pathogens from its 5'-end to 3'-end.

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