Primer sets and their use

Abstract

The application belongs to the technical field of sequencing, and particularly relates to a primer group and application thereof. The application provides a primer group, which can amplify a target library containing a normal sequencing adapter sequence through a one-step method or a two-step method. When the two-step method is used, purification of an intermediate product is not required, and the obtained amplification product has specificity and is not duplication. The primer group can be used for mNGS and / or tNGS. When the primer group is used for mNGS, the detection of a target gene can be improved. When the primer group is used for tNGS, the concentration of effective target fragments in a to-be-captured library is improved, the efficiency of pathogenic microorganism probe capture is improved, and no false positive is caused.

Description

Technical Field

[0001] This invention belongs to the field of sequencing technology, specifically relating to primer sets and their applications. Background Technology

[0002] In recent years, targeted next-generation sequencing (tNGS) of pathogenic microorganisms has gradually come into view. Among them, the targeted pathogen probe hybridization capture sequencing technology requires the extraction of nucleic acids to construct a sequencing pre-library, and then the pre-library is amplified by hybridization capture using highly specific probes designed for the target pathogen, thereby enriching the microbial sequence in the sequencing data and improving the accuracy of pathogen identification.

[0003] Compared to multiplex PCR sequencing, hybridization capture covers a wider target region. Probe design, compared to primer design, avoids interference between primers, and the maximum number of probes allowed is significantly greater than the number of primers required for multiplex PCR. Therefore, targeted pathogen probe hybridization capture sequencing technology can detect a wider variety of microbial pathogens.

[0004] Although probe hybridization capture technology can preserve low-frequency mutations of pathogens in the in vivo environment and has greater advantages for the detection of drug resistance genes, virulence genes and genomic typing of pathogens, the vast majority of human nucleic acid sequences and extremely low pathogen nucleic acid sequences in the library to be hybridized greatly limit the capture efficiency of probes for target genes such as drug resistance genes and virulence genes of pathogens in the hybridization system.

[0005] Hybrid capture has a wide coverage, but the captured pathogenic microorganisms have lower reads compared to multiplex PCR, making it unsuitable for detecting low-frequency drug resistance and virulence gene loci. Although multiplex PCR has a high depth of target region, it is often affected by primer interactions, resulting in a narrow coverage, and the reads obtained are all completely repetitive sequences, which cannot guarantee the accuracy of variant detection.

[0006] Before hybridization, PCR amplification using conventional target nucleic acids requires additional purification steps, and the resulting reads are all duplications, which cannot guarantee the accuracy of hybridization capture method for detecting variants. Summary of the Invention

[0007] The capture of pathogens by pathogen capture probes differs significantly from that of traditional probe hybridization capture. The biggest difference lies in the fact that with traditional probes, all nucleic acids in the sample have a corresponding region, meaning the probe's theoretical maximum effectiveness is 100%. However, for pathogen capture probes, the number of microorganisms infecting a sample is limited, resulting in most probes being ineffective and only a small percentage functioning. Therefore, increasing the nucleic acid concentration in the target region of the pre-read library helps the pathogen capture probe maximize its effectiveness. However, simply adding PCR to increase the nucleic acid concentration significantly increases the operation time, and the reads obtained from conventional PCR are all duplications, offering little help in improving pathogen detection in hybridization capture (hybridization capture uses reads without duplication, i.e., unique reads), to address this issue.

[0008] The first aspect of the present invention is to provide a primer set.

[0009] A second aspect of the present invention is to provide a primer-probe combination.

[0010] A third aspect of the present invention is to provide a library construction kit.

[0011] The fourth aspect of this invention is to provide a sequencing reagent kit.

[0012] The fifth aspect of this invention aims to provide a method for multiplex PCR amplification.

[0013] The sixth aspect of this invention aims to provide a method for constructing a library.

[0014] The seventh aspect of this invention is to provide a library.

[0015] The object of the eighth aspect of this invention is to provide a sequencing method.

[0016] The ninth aspect of this invention aims to provide applications of the primer set of the first aspect, the primer-probe combination of the second aspect, the library construction kit of the third aspect, and the sequencing reagent kit of the fourth aspect.

[0017] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0018] In a first aspect, the invention provides a primer set comprising: enhancing primers for amplifying a target gene;

[0019] The enhancing primer contains a universal sequence and a specific sequence from the 5' end to the 3' end; wherein...

[0020] The specific sequence is inversely complementary to at least 95%, 96%, 97%, 98%, 99%, and 100% of the target binding region of the target gene;

[0021] The universal sequence is inversely complementary to a portion or all of the sequence of the first oligonucleotide strand of the short adapter.

[0022] In some implementations, the universal sequence is reverse complementary to the 3' end sequence or the entire sequence of the first oligonucleotide strand of the short adapter.

[0023] In some implementations, the length of the universal sequence is 14-37 nt; more specifically, it is 28-32 nt.

[0024] In some embodiments, the length of the specific sequence is 12-35 nt; more specifically, it is 14-22 nt.

[0025] In some embodiments, the primer set further includes: a first universal primer and a second universal primer;

[0026] The 3' end sequence of the first universal primer is inversely complementary to the 3' end sequence of the first oligonucleotide chain;

[0027] The 3' end sequence of the second universal primer is identical to the 5' end of the second oligonucleotide strand of the short adapter.

[0028] In some embodiments, the 5' end sequence of the first oligonucleotide strand is inversely complementary to the 3' end sequence of the second oligonucleotide strand.

[0029] In some embodiments, the first oligonucleotide chain comprises, from the 5' end to the 3' end: an A' sequence and a C sequence (i.e., the universal sequence comprises, from the 5' end to the 3' end: a C' sequence or a portion of the C' sequence (preferably the 3' end sequence of the C' sequence), and an A sequence, wherein the A' sequence and the A sequence are anticomplementary, and the C sequence and the C' sequence are anticomplementary).

[0030] The second oligonucleotide chain contains, from the 5' end to the 3' end, a B sequence and an A sequence;

[0031] The A' sequence is inversely complementary to the A sequence.

[0032] In some implementations, the C sequence and the B sequence are not identical and are not reverse complementary.

[0033] In some implementations, the first universal primer comprises a sequencing primer binding sequence.

[0034] In some embodiments, the first universal primer comprises: one or more sequencing primer binding sequences; further comprising: two or more sequencing primer binding sequences.

[0035] In some implementations, the first universal primer further comprises a first tag sequence.

[0036] In some implementations, the first tag sequence is located between the sequencing primer binding sequences.

[0037] In some implementations, the first tag sequence may be a barcode sequence or an index sequence, used to distinguish different samples so that subsequent multi-sample mixed sequencing can be performed.

[0038] In some implementations, the first tag sequence may be a unique molecular tag (UMI) used to count the copy number of nucleic acid molecules in a sample.

[0039] In some implementations, the length of the first tag sequence is 5-20 nt.

[0040] In some implementations, the first universal primer further comprises a functional nucleic acid sequence.

[0041] In some embodiments, the length of the first universal primer is 30-80 nt; more specifically, it is 52-54 nt.

[0042] In some implementations, the second universal primer comprises a sequencing primer binding sequence.

[0043] In some embodiments, the second universal primer comprises: one or more sequencing primer binding sequences; further comprising: two or more sequencing primer binding sequences.

[0044] In some implementations, the second universal primer further comprises a second tag sequence.

[0045] In some embodiments, the second tag sequence is located between the sequencing primer binding sequences.

[0046] In some implementations, the second tag sequence may be a barcode sequence or an index sequence, used to distinguish different samples for subsequent multi-sample pooling sequencing.

[0047] In some implementations, the second tag sequence may be a unique molecular tag (UMI) used to count the copy number of nucleic acid molecules in a sample.

[0048] In some implementations, the length of the second tag sequence is 5-20 nt.

[0049] In some embodiments, the second universal primer further comprises a functional nucleic acid sequence.

[0050] In some embodiments, the length of the second universal primer is 40-90 nt; more specifically, it is 69-71 nt.

[0051] In some embodiments, the enhancing primers for amplifying the target gene include a variety of enhancing primers targeting different target genes.

[0052] In some embodiments, when the enhancing primers used to amplify the target gene include multiple enhancing primers targeting different target genes, any two specific sequences are not completely identical, that is, any two specific sequences may partially overlap or not overlap at all.

[0053] The primer set provided by this invention can amplify target libraries containing normal sequencing adapter sequences (long adapters) using a one-step or two-step method. In the one-step amplification method, the enhancing primer, the first universal primer, and the second universal primer are amplified simultaneously, enriching the target region without affecting the construction of the normal library. In the two-step amplification method, there is no need to purify the intermediate products. That is, after the enhancing primer pre-amplifies the target gene, the first universal primer and the second universal primer can be added directly for amplification without purifying the pre-amplification products (including the pre-amplified products and the ligation products that have not been pre-amplified). Furthermore, the excess primers after pre-amplification will not affect the subsequent library amplification. In other words, without affecting the amplification of all libraries, the overall process time is shortened and the depth and abundance of the target gene are improved.

[0054] This primer set can be used for mNGS (metagenomic next-generation sequencing) and / or tNGS (targeted next-generation sequencing):

[0055] When used in mNGS, it can improve the detection of target genes;

[0056] When used in tNGS, one type of enhancement primer can amplify multiple fragments to obtain amplification products of different fragment lengths, i.e., non-duplicative amplification products, all of which can be captured by the corresponding probe. At the same time, multiple (sets) of enhancement primers can be designed for the same probe, and the products amplified by the enhancement primers can be captured by the same probe, thereby increasing the concentration of effective target fragments in the target library, improving the efficiency of pathogenic microorganism probe capture, and avoiding false positives.

[0057] In some implementations, the primer set is used for the construction of a sample nucleic acid library, which is then used for mNGS and / or tNGS.

[0058] In some embodiments, the sample is a sample suspected of containing pathogenic microorganisms.

[0059] In some embodiments, the sample comprises at least one of cells, tissues, body fluids, saliva, urine, sputum, feces, throat swabs, and nasal swabs.

[0060] In some embodiments, the body fluid includes at least one of tissue fluid, lymph, blood, and cerebrospinal fluid.

[0061] In some embodiments, the sample is derived from an animal or plant.

[0062] In some implementations, the short adapter is an adapter in the adapter ligation product containing the fragmented genome of the sample, and a short adapter is an adapter that lacks a portion of the sequence (e.g., a tag sequence) compared to a complete sequencing adapter (long adapter).

[0063] In some embodiments, the size of the genome of the fragmented sample is 150-350 bp; more specifically, it is 200-300 bp.

[0064] In some embodiments, the target gene includes at least one of a target drug resistance gene, a virulence gene, and a pathogen gene.

[0065] In some implementations, the target gene is derived from a pathogenic microorganism.

[0066] In some embodiments, the pathogenic microorganism includes at least one of prions, fungi, bacteria, spirochetes, mycoplasma, rickettsia, chlamydia, and viruses; further, it is Mycobacterium tuberculosis or Aspergillus fumigatus.

[0067] In some embodiments, the target region is derived from Mycobacterium tuberculosis, and the sequences of the enhancing primers are shown in SEQ ID NO:1-28.

[0068] In some embodiments, when the target gene is derived from Aspergillus fumigatus, the sequence of the enhancing primer is shown in SEQ ID NO:37-56.

[0069] In some implementations, the first universal primer and the second universal primer are tag sequence primers from Geneplus's Infectious Syndrome Pathogen Nucleic Acid Targeted Detection Kit (Catalog No.: 302111202).

[0070] In some embodiments, the sequence of the first universal primer is shown in SEQ ID NO:65.

[0071] In some embodiments, the sequence of the second universal primer is shown in SEQ ID NO:64.

[0072] In this invention, the primer set with the target gene derived from Mycobacterium tuberculosis can improve the detection RPM of Mycobacterium tuberculosis in pathogen mNGS and pathogen hybrid capture tNGS technologies by adding only one step of enhanced amplification reaction time of about 20 minutes (two-step method); at the same time, without increasing the reaction time (one-step method), it can significantly improve the depth of drug resistance sites of Mycobacterium tuberculosis in pathogen hybrid capture tNGS technology; while improving the detection of Mycobacterium tuberculosis in pathogen hybrid capture tNGS technology, it will not affect the detection of other pathogens in the sample, nor will it cause false positives;

[0073] Primers targeting Aspergillus fumigatus can significantly improve the detection RPM of Aspergillus fumigatus in pathogen hybridization capture tNGS technology by adding only one step to the enhanced amplification reaction time of about 20 min (two-step method).

[0074] A second aspect of the present invention provides a primer-probe combination comprising: the primer set of the first aspect of the present invention.

[0075] In some embodiments, the primer-probe assembly further comprises: a probe;

[0076] The probe can bind to one strand of the amplification product of the enhancing primer and the second universal primer;

[0077] The binding site of the probe is located at the 5' end of the target binding region.

[0078] In some embodiments, the 3' end of the binding site of the probe is 1-X nt away from the 5' end of the target binding region, where X equals the length of the genome of the fragmented sample minus the length of the probe minus the length of the target binding region; more preferably, the 3' end of the binding site of the probe is 20-130 nt away from the 5' end of the target binding region; even more preferably, the 3' end of the binding site of the probe is 20-50 nt away from the 5' end of the target binding region of the nearest enhancing primer corresponding to the probe.

[0079] In some embodiments, the probe may bind to one strand of one of multiple amplification products (containing multiple amplification templates) of the enhancing primer and the second universal primer; and / or

[0080] The probe can bind to one strand of one of the multiple amplification products (containing an amplification template) of the multiple enhancement primers and the second universal primer.

[0081] In some embodiments, the probe is 60-150 nt in length; more specifically, it is 80-120 nt in length.

[0082] In some embodiments, the GC content of the probe is 30%-70%; more specifically, it is 45%-60%.

[0083] In some implementations, the probe comprises multiple probes.

[0084] In some implementations, when the probe includes multiple probes, no two probes are exactly the same, that is, any two probes may partially overlap or not overlap at all.

[0085] The primer-probe combination provided by this invention allows for the amplification of multiple fragments using a single enhancing primer to obtain amplification products of different fragment lengths, i.e., non-duplicative amplification products, all of which can be captured by a corresponding probe; and for the amplification of the same fragment using multiple enhancing primers to obtain amplification products at different positions, i.e., non-duplicative amplification products, which can be captured by multiple corresponding probes. Figure 3 This allows for richer fragment capture by tNGS, improved detection of unique sequences, increased capture efficiency, and more accurate identification; it can be used in tNGS.

[0086] In some implementations, the primer-probe combination is used to construct a sample nucleic acid library, which is then used for tNGS.

[0087] In some embodiments, the sample is a sample suspected of containing pathogenic microorganisms.

[0088] In some embodiments, the sample comprises at least one of cells, tissues, body fluids, saliva, urine, sputum, feces, throat swabs, and nasal swabs.

[0089] In some embodiments, the body fluid includes at least one of tissue fluid, lymph, blood, and cerebrospinal fluid.

[0090] In some embodiments, the sample is derived from an animal or plant.

[0091] In some embodiments, the pathogenic microorganism includes at least one of prions, fungi, bacteria, spirochetes, mycoplasma, rickettsia, chlamydia, and viruses; further, it is Mycobacterium tuberculosis or Aspergillus fumigatus.

[0092] In some embodiments, when the target gene is derived from Mycobacterium tuberculosis, the probe sequence is shown in SEQ ID NO:29-36.

[0093] In some embodiments, when the target gene is derived from Aspergillus fumigatus, the sequence of the probe is shown in SEQ ID NO:57-63.

[0094] A third aspect of the invention provides any one of a1)-a2) library construction kits:

[0095] a1) A library construction kit comprising: a primer set according to the first aspect of the present invention;

[0096] a2) A library construction kit comprising: a primer-probe combination according to the second aspect of the present invention.

[0097] In some implementations, the library described in a1) is an mNGS library.

[0098] In some implementations, the library described in a2) is a tNGS library.

[0099] In some embodiments, the kits described in a1) and a2) further include: a combination of nucleic acid extraction reagents.

[0100] In some embodiments, the nucleic acid extraction reagent combination is a nucleic acid extraction reagent combination selected from any of the following methods: alkaline lysis, phenol-chloroform extraction, chelating resin method, centrifugal column membrane adsorption method, and magnetic bead method; further, it is a nucleic acid extraction reagent combination for the magnetic bead method.

[0101] In some embodiments, the kits described in a1) and a2) further include reagents for breaking, end repair, adding A tails, and connector ligation.

[0102] In some embodiments, the kit described in a1) and a2) further comprises: the short connector of the first aspect of the present invention.

[0103] In some embodiments, the kit described in a2) further comprises: a reagent for hybridization capture.

[0104] A fourth aspect of the present invention provides any one of the sequencing reagent kits, b1)-b2):

[0105] b1) A sequencing reagent kit comprising: the library construction kit described in a1) of the third aspect of the present invention;

[0106] b2) A sequencing reagent kit comprising: the library construction kit described in a2) of the second aspect of the present invention.

[0107] In some embodiments, the sequencing reagent kits described in b1) and b2) further include: a sequencing kit.

[0108] In some implementations, the sequencing described in b1) is mNGS.

[0109] In some implementations, the sequencing described in b2) is tNGS.

[0110] A fifth aspect of the present invention provides a multiplex PCR amplification method comprising the step of using the primer set of the first aspect of the present invention.

[0111] In some embodiments, the multiplex PCR amplification method includes the step of performing PCR amplification on adapter ligation products of a genome containing a fragmented sample using primers from the first aspect of the invention.

[0112] In some embodiments, the PCR amplification can be a one-step PCR amplification or a two-step PCR amplification; wherein, one-step PCR amplification refers to mixing the adapter ligation product containing the fragmented sample's genome, the enhancing primer, the first universal primer, and the second universal primer, and then performing amplification; two-step PCR amplification refers to first mixing the adapter ligation product containing the fragmented sample's genome, the enhancing primer, and the second universal primer, and then performing enhanced amplification, and then mixing the enhanced amplification reaction product, the first universal primer, and the second universal primer, and then performing library amplification.

[0113] In some embodiments, the enhancement reaction product does not need to be purified during the two-step PCR amplification.

[0114] In some embodiments, the size of the genome of the fragmented sample is 150-350 bp; more specifically, it is 200-300 bp.

[0115] In some embodiments, the sample is a sample suspected of containing pathogenic microorganisms.

[0116] In some embodiments, the pathogenic microorganism includes at least one of prions, fungi, bacteria, spirochetes, mycoplasma, rickettsia, chlamydia, and viruses; further, it is Mycobacterium tuberculosis or Aspergillus fumigatus.

[0117] In some embodiments, the sample comprises at least one of cells, tissues, body fluids, saliva, urine, sputum, feces, throat swabs, and nasal swabs.

[0118] In some embodiments, the body fluid includes at least one of tissue fluid, lymph, blood, and cerebrospinal fluid.

[0119] A sixth aspect of the present invention provides a library construction method comprising the steps of the multiplex PCR amplification method of the fifth aspect of the present invention.

[0120] In some implementations, the library is an mNGS library or a tNGS library.

[0121] In some embodiments, when the library is a tNGS library, the construction method further includes the following step: using the probe of the second aspect of the present invention to hybridize and capture the multiplex PCR amplification product.

[0122] A seventh aspect of the present invention provides a library obtained by the construction method of the sixth aspect of the present invention.

[0123] An eighth aspect of the present invention provides a sequencing method for sequencing a library according to the seventh aspect of the present invention.

[0124] In some implementations, the sequencing is mNGS (when the library is an mNGS library) or tNGS (when the library is a tNGS library).

[0125] A ninth aspect of the present invention provides the use of the primer set of the first aspect, the primer-probe combination of the second aspect, the kit of the third aspect, or the sequencing reagent kit of the fourth aspect in any of c1)-c6):

[0126] c1) Construct an mNGS library or a tNGS library;

[0127] c2) Prepare products for constructing mNGS or tNGS libraries;

[0128] c3)mNGS or tNGS;

[0129] c4) Prepare products for mNGS or tNGS;

[0130] c5) Detection of pathogenic microorganisms for non-diagnostic purposes;

[0131] c6) Prepare products for detecting pathogenic microorganisms.

[0132] In some implementations, the product is a reagent kit or reagent kit.

[0133] In some embodiments, the pathogenic microorganism is the pathogenic microorganism in the first aspect of the present invention.

[0134] The beneficial effects of this invention are:

[0135] This invention provides a primer set that can amplify target libraries containing normal sequencing adapter sequences using a one-step or two-step method. The two-step amplification method eliminates the need for intermediate product purification, and the resulting amplified products are specific and non-duplicative. This primer set can be used for mNGS and / or tNGS. When used in mNGS, it improves the detection of target genes; when used in tNGS, it increases the concentration of effective target fragments in the target library, improving the efficiency of pathogen probe capture without causing false positives.

[0136] This invention provides a primer-probe combination that combines the advantages of hybridization capture and multiplex PCR. Using a single enhancing primer, multiple fragments can be amplified to obtain amplification products of different lengths, i.e., non-duplicative amplification products, all of which can be captured by a corresponding probe. Similarly, using multiple enhancing primers to amplify the same fragment yields amplification products at different positions, i.e., non-duplicative amplification products, which can also be captured by multiple corresponding probes. This enriches the fragments captured by tNGS, improves the detection of unique sequences, increases capture efficiency, and enhances identification accuracy. Furthermore, the addition of enhancing primers does not cause false positives. It can be used for tNGS. This primer-probe combination avoids the shortcomings of hybridization capture and multiplex PCR, offering short processing time, simple operation, and no additional purification steps, making it ideal for clinical pathogen detection applications. It significantly improves pathogen identification and mutation detection. Moreover, the improved detection reads are non-duplicative, resulting in a significant improvement in mutation detection accuracy. Attached Figure Description

[0137] Figure 1 The primer structure and amplified library structure of the present invention are shown: the enhancement primer (MH) is divided into two parts: part one is a specific sequence (U) of the nucleic acid of the species to be enriched, which can bind to the target binding region (U') of the target gene; part two is a fixed sequence (A-C'), wherein the conventional library adapter is (AB, A'-C), A and A' are reverse complementary, B is the same as the sequence B of primer F, and C is reverse complementary to the sequence C' of primer R. Sequence F on primer F is the fixed sequence required for sequencing, and sequence 1 is barcode 1. Sequence R on primer R is the fixed sequence at the other end required for sequencing, and sequence 2 is barcode 2. After the fixed sequence (A-C') of the enhancement primer (MH) is amplified by the library amplification primers (primer F and primer R), a normal sequencing adapter sequence can be obtained, resulting in the target library; in addition, nucleic acids that do not require enhancement can be amplified simultaneously to obtain a conventional library, and the two will not conflict during amplification. Both the target library and the conventional library can be simultaneously sequenced by mNGS or by post-hybridization sequencing (tNGS).

[0138] Figure 2 The diagram illustrates the relationship between the enhanced primers of this invention and the probe positions in tNGS: one enhanced primer can amplify multiple fragments to obtain amplification products of different fragment lengths—the amplification products that are not duplications—all of which can be captured by the corresponding probe. In the diagram, "x nt" represents the distance from the first base at the 5' end of the fragmented genome to the first base at the 5' end of the first probe (the probe closest to the 5' end), where x is an integer and x is (1, 120).

[0139] Figure 3 The diagram shows the relationship between the multiple sets of enhancement primers of this invention and the probe positions in tNGS: multiple enhancement primers can amplify the same fragment to obtain amplification products at different positions - amplification products that are not duplications - which can be captured by the corresponding multiple probes, making the fragments captured by tNGS more abundant and the identification more accurate.

[0140] Figure 4 The results of detecting Mycobacterium tuberculosis (mNGS) using the enhanced amplification technology of the present invention (enhanced amplification first, followed by library amplification) are shown: The vertical axis “Reads per M, RPM” refers to the number of reads detected by the microorganism divided by the total sequencing data (M) of nucleic acid in the sample, which is the commonly used indicator for mNGS and tNGS microbial detection, RPM (Reads per million).

[0141] Figure 5 The results of detecting Mycobacterium tuberculosis (tNGS) using the enhanced amplification technique of the present invention (enhanced amplification followed by library amplification) are shown.

[0142] Figure 6 The results of detecting Mycobacterium tuberculosis drug resistance gene loci (tNGS) using the enhanced amplification technology of the present invention (simultaneous enhanced amplification and library amplification) are shown.

[0143] Figure 7 The results of using the enhanced amplification technique of the present invention to detect the effect of Mycobacterium tuberculosis (tNGS) on the detection of other species are shown in the figure.

[0144] Figure 8 The results of detecting Aspergillus fumigatus (tNGS) using the enhanced amplification technique of the present invention are shown. Detailed Implementation

[0145] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly used in the field to which this invention pertains. For the purposes of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural forms, and vice versa.

[0146] The present invention will be further described in detail below through specific embodiments.

[0147] It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0148] Unless otherwise specified, experimental methods in the following examples are generally performed under standard conditions or as recommended by the manufacturer. Unless otherwise specified, the materials and reagents used in these examples are commercially available. For reagents whose manufacturers are listed, similar products from other manufacturers are substituted.

[0149] In this invention, the "target binding region" is located on the target gene to be enhanced. Based on the length of the region, the GC base ratio, and the base sequence specificity, the optimal binding region for amplification initiation is designed to enhance the specificity of the primer.

[0150] Example 1 Primer Design and Sequence Design

[0151] The enhancement primer (MH) of this invention consists of two parts: Part one is a specific sequence (U) of the nucleic acid of the species to be enriched, which can bind to the target binding region (U') of the target gene; Part two is a fixed sequence (A-C'), wherein the conventional library adapter is (AB, A'-C), A and A' are reverse complementary, B is the same as the sequence B of primer F, and C is reverse complementary to the sequence C' of primer R. Sequence F on primer F is the fixed sequence required for sequencing, and sequence 1 is barcode 1. Sequence R on primer R is the fixed sequence at the other end required for sequencing, and sequence 2 is barcode 2. After amplification by the library amplification primers (primer F and primer R), the fixed sequence (A-C') of the enhancement primer (MH) yields a normal sequencing adapter sequence, which can be used for normal single-strand circularization, DNB preparation, and sequencing by the MGI sequencer. More importantly, using the primers (MH) of this invention, the target nucleic acid can be amplified simultaneously with a conventional library. That is, the product of the target nucleic acid amplification can be amplified by the library amplification primers, and the primers (MH) of this invention will not conflict with the library amplification primers (F primers and R primers) during amplification. Figure 1 ).

[0152] Besides enhancing the detection of pathogens in mNGS sequencing applications, enhancement primers have a significant effect in hybridization capture tNGS technology. When the specific sequence (U) of the enhancement primer has an important positional relationship with the target sequence of the probe in tNGS technology (the 3' end of the probe binding site is 1-X nt away from the 5' end of the target binding region, where X equals the length of the fragmented sample genome - the length of the probe - the length of the target binding region, preferably 20-130 nt, more preferably the 3' end of the probe binding site is 20-50 nt away from the 5' end of the target binding region of the nearest enhancement primer corresponding to the probe, and the probe binding site is located in the genome of multiple fragmented samples), the occupancy rate of the target template can be increased during tNGS detection. Figure 2This improves the enrichment of probes in tNGS, significantly enhancing the detection of pathogenic microorganisms. Furthermore, the resulting pathogenic microorganism reads are not completely duplicated sequences, better preserving information from the original template itself, which is helpful for detecting variations in the pathogen genome, including drug resistance and virulence sites.

[0153] For illustrative purposes only, Tables 1 and 2 show the enhanced primers (C'-AU) and corresponding probe sequences for Mycobacterium tuberculosis, Aspergillus fumigatus and their resistance sites.

[0154] Table 1. Primers and corresponding probe sequences for enhancing Mycobacterium tuberculosis and its drug resistance sites.

[0155]

[0156]

[0157]

[0158] Note: In the above enhancement primers, the bolded part is the fixed sequence: the single underlined part is C', and the double underlined part is A; the unbolded part is the specific sequence (U).

[0159] Table 2. Enhanced primers and corresponding probe sequences for Aspergillus fumigatus detection.

[0160]

[0161]

[0162] Note: In the above enhancement primers, the bolded part is the fixed sequence: the single underlined part is C', and the double underlined part is A; the unbolded part is the specific sequence (U).

[0163] The library primers F and R used in the following examples are tag sequence primers from Geneplus's "Infectious Syndrome Pathogen Nucleic Acid Targeted Detection Kit" (Catalog No.: 302111202). The sequence of library primer F is: 5'p-TCTCAGTACGTCAGCAGT TNNNNNNNNNNCAACTCCTTGGCTCACAGAACGACATGGCTACGATCCGACT (SEQ ID NO: 64), where NNNNNNNNNN is the tag sequence and p is phosphorylation modification. The sequence of library primer R is: GGCATGGCGACCTTATCAGNN NNNNNNNNTTGTCTTCCTAAGACCGCTTGGCC (SEQ ID NO: 65), where NNNNNNNNNN is the tag sequence and N is independently selected from A, T, C, and G.

[0164] Example 2: Enhanced detection of Mycobacterium tuberculosis (first enhanced amplification, then library amplification)

[0165] This embodiment uses the detection of Mycobacterium tuberculosis in a standard sample via sequencing as an example to illustrate the performance of the technology of the present invention. The specific steps are as follows:

[0166] 1. Sample preparation: Add 10 μL of PBS buffer to 1 mL of PBS buffer. 5 Cells A549 cell line, prepared to a concentration of 1*10 5 Human nucleic acid background solution with concentrations of cells / mL was prepared, and then Mycobacterium tuberculosis was added to the prepared human nucleic acid background solution at final concentrations of 50 copies / mL, 500 copies / mL, and 5000 copies / mL, respectively, with no Mycobacterium tuberculosis added (0 copies / mL) as a control to obtain the test standard.

[0167] 2. Nucleic acid extraction: Take 400 μL of the standard from step 1 and use Geneplus's Pathogenic Microorganism DNA / RNA Extraction Kit (Magnetic Bead Method) (Catalog No.: KB0055048) to extract nucleic acid. The extracted product is quantified using Qubit.

[0168] 3. Library Construction: 25 ng of quantified DNA was used to construct a library using the Geneplus Infectious Disease Syndrome Pathogen Nucleic Acid Targeted Detection Kit (Catalog No.: 302111202). The steps included nucleic acid fragmentation, end repair and A-tailing, adapter ligation, and magnetic bead purification. The purified product after adapter ligation was reconstituted in 22 μL of H2O.

[0169] 4. Enhanced amplification of this invention

[0170] 4.1 Enhanced Amplification

[0171] The amplification system was prepared according to the following composition (Table 3). The total reaction volume was 48 μL. The enhanced amplification reaction was run in the PCR instrument according to the following program: 98℃ for 1 min; (98℃ for 15 s, 55℃ for 30 s, 72℃ for 30 s) for 9 cycles; 72℃ for 5 min; 4℃ hold.

[0172] Table 3

[0173]

[0174] Note: Primer F for the library is from Geneplus's "Infectious Syndrome Pathogen Nucleic Acid Targeted Detection Kit" (Catalog No.: 302111202).

[0175] 4.2 Library amplification

[0176] After enhanced amplification, place the amplification product on ice and immediately add the library amplification primers listed in Table 4. Library amplification can then be performed directly (no purification required). Library primers F and R are from Geneplus's "Infectious Syndrome Pathogen Nucleic Acid Targeted Detection Kit" (catalog number: 302111202). The total reaction volume is 50 μL. Run the library amplification reaction in the PCR instrument according to the following procedure: 98℃ for 1 min; (98℃ for 15 s, 60℃ for 30 s, 72℃ for 30 s) for 8 cycles; 72℃ for 5 min; 4℃ hold.

[0177] Table 4

[0178]

[0179] After amplification, 45 μL of magnetic beads were added to the amplification product for magnetic bead purification. The purified product was then reconstituted in 30 μL of H2O. The obtained product was quantified using a quorum analyzer. Part of the product was used for mNGS sequencing (directly for 5.6), and the other part was used for pathogen probe hybridization reaction (tNGS).

[0180] 5. Hybridization capture was performed using the pathogen capture reagent, probe, and amplification product from step 4.2 of the Geneplus "Infectious Syndrome Pathogen Nucleic Acid Targeted Detection Kit" (catalog number: 302111202). The probe contains the probe sequence corresponding to the present invention (Table 1). The specific steps are as follows:

[0181] 5.1 Preparation of hybridization system

[0182] Take 1200 ng of the amplification product from step 4.2 and prepare the nucleic acid hybridization system in a 1.5 mL centrifuge tube according to the table below (Table 5). Use a vacuum concentrator to evaporate the prepared nucleic acid to dryness at 60 °C.

[0183] Table 5

[0184]

[0185]

[0186] 5.2 Hybridization reaction

[0187] Prepare the rapid hybridization reaction solution according to Table 6. After mixing the prepared hybridization buffer, add it to the evaporated nucleic acid to be hybridized in step 5.1. Incubate at room temperature for 10 min, then transfer the liquid to a 0.2 mL low-adsorption centrifuge tube and add 4 μL of Geneplus pathogen hybridization probe. Mix well and perform the rapid hybridization reaction according to the following procedure: 95℃, 10 min; 65℃, 1 h.

[0188] Table 6

[0189]

[0190] 5.3 Hybridization of magnetic beads

[0191] 5.3.1. Take 10 μL of M270 Streptavidin magnetic beads (magnetic beads B), clean them with 1X magnetic bead cleaning solution, and discard the supernatant.

[0192] 5.3.2. Transfer the hybridization reaction product to magnetic beads and incubate at 65°C for 45 min using a PCR instrument.

[0193] 5.3.3. Vortex mix for 3 seconds every 9 minutes to ensure the magnetic beads are in a suspended state.

[0194] 5.4 Washing after hybridization

[0195] 5.4.1 Hot cleaning

[0196] Add 100 μL of 1X washing solution I (preheated to 65°C) to the magnetic bead mixture containing the hybridization reaction product, mix well, and place on a magnetic rack to remove the supernatant. Wash twice with 200 μL of preheated 1X washing solution S, and remove the supernatant after each wash.

[0197] 5.4.2 Room temperature cleaning

[0198] Clean the magnetic beads once each in sequence using 180 μL of 1X Cleaning Solution I, 180 μL of 1X Cleaning Solution II, and 180 μL of 1X Cleaning Solution III. After cleaning, resuspend the magnetic beads in 20 μL of Nuclease-free water.

[0199] 5.5 Amplification after hybridization:

[0200] Add 25 μL of capture product polymerase mixture and 4 μL of amplification primers (20 μM, universal primers required for the sequencing platform, from the "Infectious Syndrome Pathogen Nucleic Acid Targeted Detection Kit" (catalog number: 302111202)) to the system after washing in step 5.4. Mix thoroughly and perform cyclic amplification: 98℃ for 1 min; 98℃ for 10 s, 60℃ for 30 s, 72℃ for 30 s (8 cycles); 72℃ for 1 min; 4℃ hold. After amplification, add 50 μL of magnetic beads to the amplified product for magnetic bead purification. The purified product is then reconstituted in 30 μL of TE.

[0201] 5.6 Sequencing and Data Analysis

[0202] The library amplified in step 5.5 was prepared into DNB using the MGI single-strand circularization kit and the MGISEQ-G99 high-throughput sequencing kit (SE100), and then sequenced using the MGISE-G99 sequencer with SE100 sequencing. Sequencing results were compared and analyzed against pathogen databases using BWA.

[0203] This embodiment uses conventional library construction and hybridization capture without the enhancement method of this invention as a control. Specifically, the control group used the same nucleic acid extraction products for Geneplus's "Infectious Syndrome Pathogen Nucleic Acid Targeted Detection Kit" (catalog number: 302111202) mNGS library construction and tNGS hybridization capture followed by sequencing. The amount of pre-sequencing data was consistent with the experimental group using the enhancement amplification method of this invention (i.e., the control group differs from this embodiment in that it does not include "4.1 Enhancement Amplification," and directly uses the purified product after adapter ligation as a template for "4.2 Library Amplification"). The reaction system for library amplification in the control group is shown in Table 7. The PCR program was: 98℃ for 1 min; (98℃ for 15 s, 60℃ for 30 s, 72℃ for 30 s) for 9 cycles; 72℃ for 5 min; 4℃ hold. The total reaction volume was 50 μL.

[0204] Table 7

[0205]

[0206] The results are as follows Figure 4 , 5 As shown, the enhanced amplification technology of this invention (enhanced amplification first, followed by library amplification) can significantly improve the detection of Mycobacterium tuberculosis in pathogen mNGS and pathogen hybridization capture tNGS technologies without causing false positives.

[0207] Example 3: Enhancing the depth of drug resistance sites in Mycobacterium tuberculosis (simultaneous enhancement amplification and library amplification)

[0208] This embodiment uses the sequencing detection of the depth of drug resistance sites in Mycobacterium tuberculosis in a standard sample as an example to illustrate the performance of the technology of the present invention. The method of this embodiment is the same as that of Embodiment 2, except for "4. Enhanced amplification of the present invention", which is as follows:

[0209] The amplification system was prepared according to the following grouping (Table 8). The total reaction volume was 50 μL. The library amplification reaction was run in the PCR instrument according to the following program: 98℃ for 1 min; (98℃ for 15 s, 60℃ for 30 s, 72℃ for 30 s) for 13 cycles; 72℃ for 5 min; 4℃ hold. After amplification, 45 μL of magnetic beads were added to the amplified product for magnetic bead purification, and the purified product was reconstituted in 30 μL of H2O. The obtained product was quantified using a Qubit analyzer. The obtained product was used for pathogen probe hybridization reaction (method as in Example 2).

[0210] Table 8

[0211]

[0212] In this embodiment, the tNGS sequencing results after conventional library construction and hybridization capture of the same nucleic acid extraction product without using the enhancement method of the present invention were used as a control (the method is the same as in Example 2).

[0213] The results are as follows Figure 6 As shown: Using the enhanced amplification technology of the present invention (enhanced amplification and library amplification are performed simultaneously), the RPM of the sequences of 33 drug resistance gene loci of Mycobacterium tuberculosis in pathogen hybridization capture tNGS technology can be significantly improved.

[0214] Example 4: Effect of the Mycobacterium tuberculosis enhancement primers of the present invention on the detection of other species

[0215] This embodiment uses the detection of pathogenic microorganisms in a standard sample via sequencing as an example to illustrate the performance of the present invention in improving the detection of mycobacteria without affecting the detection of other pathogenic microorganisms. The specific steps are the same as in Example 2, except for sample preparation. The sample preparation steps are as follows:

[0216] Sample preparation: Add 1x 10 to 1 mL of PBS buffer. 5 Cells A549 cell line, prepared at a concentration of 1x10⁻⁶. 5 Human nucleic acid background solution was prepared by adding 50 copies / mL of Escherichia coli, 50 copies / mL of Staphylococcus aureus, 50 copies / mL of Enterococcus faecalis, 100 copies / mL of Pseudomonas aeruginosa, 100 copies / mL of Listeria monocytogenes, 100 copies / mL of Salmonella enterica, and 100 copies / mL of Mycobacterium tuberculosis complex to the prepared human nucleic acid background solution to prepare standard samples.

[0217] In this embodiment, the tNGS sequencing results after conventional library construction and hybridization capture of the same nucleic acid extraction product without using the enhancement method of the present invention were used as controls - "100 copies / mL tuberculosis" (method is the same as in Example 2); control (0 copies / mL tuberculosis) (method is the same as in Example 2, but the sample does not contain Mycobacterium tuberculosis).

[0218] The results are as follows Figure 7 As shown: The enhanced amplification technology of the present invention can improve the detection of Mycobacterium tuberculosis in pathogen hybridization capture tNGS technology without affecting the detection of other pathogens in the sample or causing false positives.

[0219] Example 5: Improved detection of Aspergillus fumigatus

[0220] The specific steps in this embodiment are the same as in Example 2, except for the differences in sample preparation and the use of enhancement primers. The sample preparation steps are as follows:

[0221] Sample preparation: Add 1x 10 to 1 mL of PBS buffer. 5 Cells A549 cell line, prepared at a concentration of 1x10⁻⁶. 5 A human nucleic acid background solution with a concentration of cells / mL was prepared, and then Aspergillus fumigatus with a final concentration of 100 CFU / mL was added to the prepared human nucleic acid background solution to prepare a standard sample.

[0222] The enhancing primers were the Aspergillus fumigatus enhancing primers listed in Table 2. The Geneplus Pathogenic Microorganism Targeted High-Throughput Gene Detection Kit (Catalog No.: KB0032048) used contained the probe sequences corresponding to the present invention (Table 2).

[0223] This embodiment uses conventional library construction and hybridization capture without using the enhancement method of the present invention as a control (the method is the same as in Example 2).

[0224] The results are as follows Figure 8 As shown: Using the enhanced amplification technology of the present invention, the detection RPM of Aspergillus fumigatus in pathogen hybridization capture tNGS technology can be significantly improved.

[0225] The technical solutions of the present invention are not limited to the specific embodiments described above. Any technical modifications made in accordance with the technical solutions of the present invention fall within the protection scope of the present invention.

Claims

1. A primer set, characterized in that, The primer set includes: a primer set for enhancing primers used to amplify the target gene; When the target gene is derived from Mycobacterium tuberculosis, the primer set of the enhancing primers consists of a set of primers as shown in SEQ ID NO:1-28; and / or When the target gene is derived from Aspergillus fumigatus, the primer set of the enhancing primers is a set of primers as shown in SEQ ID NO:37-56.

2. The primer set according to claim 1, characterized in that, The primer set further includes: a first universal primer and a second universal primer; The 3' end sequence of the first universal primer is inversely complementary to the 3' end sequence of the first oligonucleotide strand of the short adapter; The 3' end sequence of the second universal primer is identical to the 5' end sequence of the second oligonucleotide strand of the short adapter; The 5' end sequence of the first oligonucleotide chain is inversely complementary to the 3' end sequence of the second oligonucleotide chain.

3. The primer set according to claim 2, characterized in that, The first oligonucleotide chain contains, from the 5' end to the 3' end, an A' sequence and a C sequence; The second oligonucleotide chain contains, from the 5' end to the 3' end, a B sequence and an A sequence; The A' sequence is inversely complementary to the A sequence; and / or The first universal primer comprises: a sequencing primer binding sequence; and / or The first universal primer further comprises: a first tag sequence; and / or The second universal primer comprises: a sequencing primer binding sequence; and / or The second universal primer also includes a second tag sequence.

4. The primer set according to claim 3, characterized in that, The first label sequence is a barcode sequence or an index sequence; and / or The second label sequence is a barcode sequence or an index sequence; and / or The sequence of the first universal primer is shown in SEQ ID NO:65; and / or The sequence of the second universal primer is shown in SEQ ID NO:

64.

5. A primer-probe combination, characterized in that, The combination comprises: the primer set and probe as described in any one of claims 1-4.

6. The primer-probe combination according to claim 5, characterized in that, The probe binds to one strand of the amplification product of the enhancing primer and the second universal primer; The binding site of the probe is located at the 5' end of the target binding region.

7. The primer-probe combination according to claim 6, characterized in that, The 3' end of the probe's binding site is 1-Xnt from the 5' end of the target binding region, where X equals the length of the fragmented sample's genome - the length of the probe - the length of the target binding region.

8. The primer-probe combination according to claim 7, characterized in that, The probe is 60-150 nt in length.

9. The primer-probe combination according to claim 8, characterized in that, When the target gene is derived from Mycobacterium tuberculosis, the probe sequence is as shown in SEQ ID NO:29-36; and / or When the target gene is derived from Aspergillus fumigatus, the probe sequence is shown in SEQ ID NO:57-63.

10. A kit for constructing any type of library (a1)-a2): a1) A library construction kit comprising: the primer set according to any one of claims 1-4; a2) A library construction kit comprising: the primer-probe combination as described in any one of claims 5-9.

11. The library construction kit according to claim 10, characterized in that, a1) The library mentioned is an mNGS library; a2) The library mentioned is a tNGS library; and / or The kits described in a1) and a2) also include: a combination of nucleic acid extraction reagents.

12. The library construction kit according to any one of claims 10-11, characterized in that, The kits described in a1) and a2) also contain reagents for breaking, end repair, adding A tails, and connector ligation.

13. The library construction kit according to claim 12, characterized in that, a2) The kit also contains: reagents for hybridization capture. 14.b1)-b2) Any one of the sequencing reagent kits: b1) A sequencing reagent kit comprising: the library construction kit described in a1) of any one of claims 10-13; b2) A sequencing reagent kit comprising: the library construction kit as described in a2) of any one of claims 10-13.

15. The sequencing reagent kit according to claim 14, characterized in that, The sequencing reagent kits described in b1) and b2) also include: a sequencing kit; and / or b1) The sequencing described is mNGS; b2) The sequencing described is tNGS.

16. A multiplex PCR amplification method, characterized in that... The multiplex PCR amplification method comprises the following steps: performing PCR amplification on the adapter ligation product of the genome containing the fragmented sample using the primer set according to any one of claims 1-4.

17. The multiplex PCR amplification method according to claim 16, characterized in that, The PCR amplification is either a one-step PCR amplification or a two-step PCR amplification.

18. The multiplex PCR amplification method according to claim 17, characterized in that, One-step PCR amplification refers to the process of mixing adapter ligation products containing the fragmented genome of a sample, enhancing primers, a first universal primer, and a second universal primer, and then performing amplification; and / or Two-step PCR amplification involves first mixing the adapter ligation product containing the fragmented genome of the sample, the enhancing primer, and the second universal primer for enhanced amplification; then mixing the enhanced amplification reaction product, the first universal primer, and the second universal primer for library amplification.

19. The multiplex PCR amplification method according to claim 18, characterized in that, The two-step PCR amplification method does not require purification of the enhancement reaction product.

20. A library construction method, characterized in that, The method comprises the steps of the multiplex PCR amplification method according to any one of claims 16-19.

21. The library construction method according to claim 20, characterized in that, The library is an mNGS library or a tNGS library.

22. The library construction method according to claim 21, characterized in that, When the library is a tNGS library, the construction method further includes the following step: using the probes in the primer-probe combination described in any one of claims 5-9 to hybridize and capture the multiplex PCR amplification product.

23. A library, characterized in that, The library is obtained by the library construction method according to any one of claims 20-22.

24. A sequencing method, characterized in that, The method includes sequencing the library of claim 23.

25. The sequencing method according to claim 24, characterized in that, The sequencing is mNGS or tNGS.

26. The use of the primer set of any one of claims 1-4, the primer-probe combination of any one of claims 5-9, the kit of any one of claims 10-13, or the sequencing reagent kit of any one of claims 14-15 in any one of c1)-c6): c1) is used to construct mNGS or tNGS libraries; c2) Prepare products for constructing mNGS or tNGS libraries; c3) is used for non-diagnostic destination mNGS sequencing or non-diagnostic destination tNGS sequencing; c4) Prepare products for mNGS or tNGS; c5) Used for non-diagnostic detection of pathogenic microorganisms; c6) Prepare products for detecting pathogenic microorganisms; The pathogenic microorganisms are Mycobacterium tuberculosis and / or Aspergillus fumigatus.

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