Primer group for detecting free nucleic acid of bloodstream infection pathogen, kit and application

Abstract

The invention discloses a primer group for detecting free nucleic acid of a blood flow infection pathogen, a kit and application. The primer group comprises 19 groups of primers, trace and fragmented pathogen cfDNA in a blood sample can be subjected to targeted linear enrichment through the primer group, and then synchronous detection of multiple pathogens and drug-resistant genes is performed in combination with high-throughput sequencing. And finally, synchronous identification of 15 clinical important bloodstream infection pathogens and 4 drug-resistant genes can be realized within a relatively short time (about 12 hours), the method has high sensitivity and high specificity, the detection limit can reach 0.1 copy / mL, the detection cost and the operation difficulty are greatly reduced, and the method is suitable for clinical popularization and application.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a primer set, reagent kit, and application for detecting free nucleic acids of bloodstream infection pathogens. Background Technology

[0002] Bloodstream infection (BSI) is a serious infectious disease caused by pathogenic microorganisms invading the bloodstream. It can rapidly develop into sepsis, septic shock, and even be life-threatening. Timely and accurate diagnosis of bloodstream infection pathogens and determination of their drug resistance are crucial for guiding clinical treatment and improving patient prognosis.

[0003] At present, the gold standard for diagnosing bloodstream infections is blood culture, which identifies the pathogen through biochemical methods or microscopic observation. However, this method has the following shortcomings: (1) The culture cycle is long, usually requiring 2-3 days or even longer to determine the result, which cannot meet the needs of rapid clinical diagnosis; (2) The sensitivity and positive rate are low, especially for low-concentration infections and infections with special pathogens (such as fungi and anaerobic bacteria), which are prone to false negative results; (3) It cannot directly provide information on the drug resistance of the pathogen, and subsequent drug susceptibility testing is required.

[0004] To break free from the constraints of traditional technologies, molecular biology techniques have been increasingly incorporated into clinical medicine beyond general scientific research in recent years. Examples include traditional PCR / qPCR / digital PCR methods and metagenomic sequencing (mNGS). While these methods have improved detection speed and sensitivity to some extent, they still present the following challenges: PCR targets the nucleic acids of pathogens rather than live bacteria. For routine clinical specimens with randomly fragmented nucleic acid molecules, such as cell-free DNA (cfDNA) in plasma and urine, the sensitivity of PCR detection is limited by the ratio of the length of the target nucleic acid molecule to the length of the PCR amplicon. In detection, it is usually difficult to effectively amplify short fragments (around 40-100 bp) of cfDNA, resulting in low sensitivity. mNGS can theoretically achieve unbiased pathogen detection, but its sequencing and data analysis costs are high, and it is also inefficient in detecting short fragment cfDNA: cfDNA fragments in blood samples are short (usually 40-100 bp), and the content of pathogen cfDNA is extremely low (usually less than 1% of total cfDNA). mNGS is prone to losing these short fragment molecules during library construction, with a molecular conversion rate of only 10%-30%, which cannot efficiently capture short fragment target molecules, resulting in decreased detection sensitivity.

[0005] Therefore, how to effectively detect trace amounts of pathogen cfDNA in blood, so as to quickly, sensitively and accurately diagnose clinical bloodstream infections, is a technical problem that urgently needs to be solved in this field. Summary of the Invention

[0006] The purpose of this invention is to provide a primer set, kit, and application for detecting cell-free nucleic acids of bloodstream pathogens. This invention enables targeted linear enrichment of trace, fragmented pathogen cfDNA in blood samples, combined with high-throughput sequencing for simultaneous detection of multiplex pathogens and drug resistance genes. Ultimately, it achieves simultaneous identification of 15 clinically important bloodstream pathogens and 4 drug resistance genes within a short time (approximately 12 hours), exhibiting high sensitivity and specificity, with a detection limit as low as 0.1 copies / mL. Furthermore, it significantly reduces detection costs and operational complexity, making it suitable for clinical promotion and application.

[0007] To achieve the above objectives, the present invention first provides a primer set for detecting cell-free nucleic acids of bloodstream infection pathogens, the primer set comprising the following 19 primer groups:

[0008] (1) Primers for detecting free nucleic acid of Acinetobacter baumannii, the sequences of which are any one or more of SEQ ID NO: 1 to SEQ ID NO: 3;

[0009] (2) Primers for detecting free nucleic acid of Candida albicans, the sequences of which are any one or more of SEQ ID NO: 4 to SEQ ID NO: 6;

[0010] (3) Primers for detecting free nucleic acid of Candida glabrata, the sequences of which are any one or more of SEQ ID NO: 7 to SEQ ID NO: 9;

[0011] (4) Primers for detecting free nucleic acid of Enterobacter cloacae, the sequences of which are any one or more of SEQ ID NO: 10 to SEQ ID NO: 12;

[0012] (5) Primers for detecting Enterococcus faecalis free nucleic acid, the sequences of which are any one or more of SEQ ID NO: 13 to SEQ ID NO: 15;

[0013] (6) Primers for detecting Enterococcus faecalis free nucleic acid, the sequences of which are any one or more of SEQ ID NO: 16 to SEQ ID NO: 18;

[0014] (7) Primers for detecting free nucleic acid of Escherichia coli, the sequences of which are any one or more of SEQ ID NO: 19 to SEQ ID NO: 21;

[0015] (8) Primers for detecting Klebsiella pneumoniae free nucleic acid, the sequences of which are any one or more of SEQ ID NO: 22 to SEQ ID NO: 24;

[0016] (9) Primers for detecting free nucleic acid of Proteus mirabilis, having the sequence of any one or more of SEQ ID NO: 25 to SEQ ID NO: 27;

[0017] (10) Primers for detecting free nucleic acid of Pseudomonas aeruginosa, the sequences of which are any one or more of SEQ ID NO: 28 to SEQ ID NO: 30;

[0018] (11) Primers for detecting Serratia marcescens free nucleic acid, the sequences of which are any one or more of SEQ ID NO: 31 to SEQ ID NO: 33;

[0019] (12) Primers for detecting free nucleic acid of Staphylococcus aureus, the sequences of which are any one or more of SEQ ID NO: 34 to SEQ ID NO: 36;

[0020] (13) Primers for detecting free nucleic acid of Staphylococcus epidermidis, having the sequence of any one or more of SEQ ID NO: 37 to SEQ ID NO: 39;

[0021] (14) Primers for detecting free nucleic acid of Streptococcus pyogenes, having the sequence of any one or more of SEQ ID NO: 40 to SEQ ID NO: 42;

[0022] (15) Primers for detecting free nucleic acid of Streptococcus pneumoniae, the sequences of which are any one or more of SEQ ID NO: 43 to SEQ ID NO: 45.

[0023] (16) Primers for detecting the drug resistance gene KPC, the sequences of which are any one or more of SEQ ID NO: 46 to SEQ ID NO: 48;

[0024] (17) Primers for detecting the drug resistance gene mecA, the sequences of which are any one or more of SEQ ID NO: 49 to SEQ ID NO: 51;

[0025] (18) Primers for detecting the drug resistance gene NDM, the sequences of which are any one or more of SEQ ID NO: 52 to SEQ ID NO: 54;

[0026] (19) Primers for detecting the drug resistance gene OXA-48, the sequences of which are any one or more of SEQ ID NO: 55 to SEQ ID NO: 57.

[0027] Preferably, each primer has the following dual modifications: (1) thiomodification: the phospholipid bonds of the 3' end portion of the nucleotide backbone of the primer are thiomodified; (2) blocking modification: the 3' end of the primer has a blocking modification to prevent direct extension.

[0028] Preferably, the thiomodification refers to the conversion of the phospholipid bonds in the nucleotide backbone from double bonds O to double bonds S.

[0029] Preferably, the blocking modification is: the hydroxyl group at the 3-position of the nucleotide at the 3' end of the primer is replaced by a dimorphic functional group.

[0030] Preferably, the dimorphic functional group includes any one or more of the following: phosphate group, biotin group, C6 spacer group, and NH2-C6 group.

[0031] In another aspect, the present invention provides a kit for detecting cell-free nucleic acids of bloodstream infection pathogens, the kit comprising the aforementioned primer set.

[0032] Preferably, the kit further comprises at least one of the following: plasma nucleic acid cell-free DNA extraction reagent, linear amplification reagent, adapter ligation reagent, or library construction reagent.

[0033] Preferably, the linear amplification reagent comprises DNA polymerase, dNTPs, and PCR amplification buffer.

[0034] Preferably, the DNA polymerase has 3'-5' exonuclease activity.

[0035] In another aspect, the present invention provides the application of the aforementioned kit in the detection of free nucleic acids of bloodstream infection pathogens.

[0036] The present invention has at least the following beneficial effects:

[0037] This invention is the first to successfully apply OPERA technology to the detection of cell-free nucleic acids of bloodstream pathogens. Specifically, by using a specific single primer (SEQ ID NO: 1 to SEQ ID NO: 57) in conjunction with DNA polymerase (with 3'-5' exonuclease activity), it is possible to target and linearly enrich trace amounts of fragmented pathogen cfDNA in blood samples. Combined with high-throughput sequencing, multiplex pathogens and drug resistance genes can be detected simultaneously. Ultimately, it can achieve the simultaneous identification of 15 clinically important bloodstream pathogens and 4 drug resistance genes in a relatively short time (approximately 12 hours). It has high sensitivity and high specificity, and can accurately detect even samples with low pathogen load. The detection limit can reach 0.1 copies / mL, and the detection cost and operation difficulty are significantly reduced, making it suitable for clinical promotion and application. Attached Figure Description

[0038] Figure 1A-1O The results represent the sensitivity analysis of reference materials for detecting pathogens such as Escherichia coli, Enterobacter cloacae, Klebsiella pneumoniae, Serratia marcescens, Pseudomonas aeruginosa, Acinetobacter baumannii, Enterococcus faecalis, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pneumoniae, Streptococcus pyogenes, Candida albicans, Candida glabrata, and Proteus mirabilis. Detailed Implementation

[0039] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0040] It should be noted that the following detailed descriptions are exemplary and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0041] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments according to this application. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0042] As mentioned earlier, existing bloodstream infection (BSI) detection methods (such as blood culture, PCR / qPCR / ddPCR, mNGS, etc.) suffer from problems such as slow speed, low sensitivity, narrow coverage, large background interference, and high cost when detecting trace amounts of fragmented pathogen cfDNA in blood samples. Clinically, there is an urgent need for a detection solution that can rapidly, sensitively, accurately, and with multiplex detection of trace pathogen cfDNA in blood.

[0043] To address the aforementioned technical problems, this invention, based on previous research into various amplification technologies, recognizes the potential of single-primer linear amplification (OPERA) technology in handling low starting amounts and fragmented DNA. Theoretically, linear amplification better preserves the relative abundance information of the original sample and avoids the bias problems caused by exponential amplification. Therefore, OPERA technology is applied for the first time to the detection of fragmented pathogenic nucleic acid molecules mcfDNA in blood. Through primer sequence design and screening, and optimization of primer working concentration, a kit for detecting cell-free nucleic acids of bloodstream pathogens is finally provided. This kit contains: specific single primers, DNA polymerase, and dNTPs. By using a specific single primer in conjunction with DNA polymerase, trace amounts and fragmented pathogen cfDNA in blood samples can be linearly enriched. This can then be combined with high-throughput sequencing for simultaneous detection of multiple pathogens and drug resistance genes. Ultimately, this method can simultaneously identify 15 clinically important bloodstream infection pathogens and 4 drug resistance genes within a short time (approximately 12 hours). It has high sensitivity and high specificity, and can accurately detect even low pathogen load samples. Furthermore, it significantly reduces detection costs and operational difficulty, making it suitable for clinical promotion and application.

[0044] The specific single primers include the following 19 sets of primers:

[0045] (1) Primers for detecting free nucleic acid of Acinetobacter baumannii, the sequences of which are any one or more of SEQ ID NO: 1 to SEQ ID NO: 3;

[0046] (2) Primers for detecting free nucleic acid of Candida albicans, the sequences of which are any one or more of SEQ ID NO: 4 to SEQ ID NO: 6;

[0047] (3) Primers for detecting free nucleic acid of Candida glabrata, the sequences of which are any one or more of SEQ ID NO: 7 to SEQ ID NO: 9;

[0048] (4) Primers for detecting free nucleic acid of Enterobacter cloacae, the sequences of which are any one or more of SEQ ID NO: 10 to SEQ ID NO: 12;

[0049] (5) Primers for detecting Enterococcus faecalis free nucleic acid, the sequences of which are any one or more of SEQ ID NO: 13 to SEQ ID NO: 15;

[0050] (6) Primers for detecting Enterococcus faecalis free nucleic acid, the sequences of which are any one or more of SEQ ID NO: 16 to SEQ ID NO: 18;

[0051] (7) Primers for detecting free nucleic acid of Escherichia coli, the sequences of which are any one or more of SEQ ID NO: 19 to SEQ ID NO: 21;

[0052] (8) Primers for detecting Klebsiella pneumoniae free nucleic acid, the sequences of which are any one or more of SEQ ID NO: 22 to SEQ ID NO: 24;

[0053] (9) Primers for detecting free nucleic acid of Proteus mirabilis, having the sequence of any one or more of SEQ ID NO: 25 to SEQ ID NO: 27;

[0054] (10) Primers for detecting free nucleic acid of Pseudomonas aeruginosa, the sequences of which are any one or more of SEQ ID NO: 28 to SEQ ID NO: 30;

[0055] (11) Primers for detecting Serratia marcescens free nucleic acid, the sequences of which are any one or more of SEQ ID NO: 31 to SEQ ID NO: 33;

[0056] (12) Primers for detecting free nucleic acid of Staphylococcus aureus, the sequences of which are any one or more of SEQ ID NO: 34 to SEQ ID NO: 36;

[0057] (13) Primers for detecting free nucleic acid of Staphylococcus epidermidis, having the sequence of any one or more of SEQ ID NO: 37 to SEQ ID NO: 39;

[0058] (14) Primers for detecting free nucleic acid of Streptococcus pyogenes, having the sequence of any one or more of SEQ ID NO: 40 to SEQ ID NO: 42;

[0059] (15) Primers for detecting free nucleic acid of Streptococcus pneumoniae, the sequences of which are any one or more of SEQ ID NO: 43 to SEQ ID NO: 45.

[0060] (16) Primers for detecting the drug resistance gene KPC, the sequences of which are any one or more of SEQ ID NO: 46 to SEQ ID NO: 48;

[0061] (17) Primers for detecting the drug resistance gene mecA, the sequences of which are any one or more of SEQ ID NO: 49 to SEQ ID NO: 51;

[0062] (18) Primers for detecting the drug resistance gene NDM, the sequences of which are any one or more of SEQ ID NO: 52 to SEQ ID NO: 54;

[0063] (19) Primers for detecting the drug resistance gene OXA-48, the sequences of which are any one or more of SEQ ID NO: 55 to SEQ ID NO: 57.

[0064] The specific sequences of the above-mentioned specific single primers are detailed in Table 11 below. Each primer contains a general sequencing sequence and a specific sequence from the 5' end to the 3' end. The primers have the following dual modifications: (1) Thio modification: The phospholipid bonds of the nucleotide backbone at the 3' end of the primer are thio modified, specifically, the phospholipid bonds of the nucleotide backbone are changed from double bonds O to double bonds S, which is used to reduce primer dimers and non-specific amplification during linear amplification and improve detection specificity; (2) Blocking modification: The 3' end of the primer has a blocking modification. This modification is used on the one hand to prevent direct extension: After the primer binds to the target region, it cannot undergo an extension reaction under the conventional DNA polymerase conditions. Instead, it needs to be recognized and activated by a DNA polymerase with primer activation function before the linear amplification reaction can be started; on the other hand, it is used to prevent direct ligation: It prevents the primer (especially the free primer that has not bound to the target region) from being directly and non-specifically ligated to the adapter in subsequent steps, ensuring the accuracy of library construction. As a specific example, the blocking modification specifically refers to the substitution of the 3-C hydroxyl group at the 3' end of the primer with a dimorphic functional group, which can be a phosphate group, a biotin group, a C6 spacer group, an NH2-C6 group, etc.

[0065] The primer-activating DNA polymerase possesses 3'-5' exonuclease activity. This enzyme recognizes a specific single primer that binds to the template (i.e., the target region), then removes the blocking modification group at the 3' end of the template-binding primer, thereby activating the primer and enabling it to initiate the subsequent linear extension reaction. This ensures that only primers that correctly bind to the template sequence are activated and amplified, further enhancing specificity. As a specific example, the primer-activating DNA polymerase is a group B DNA polymerase.

[0066] In this invention, after targeted linear amplification of trace, fragmented pathogenic cfDNA in blood samples using the aforementioned dual-modified specific single primer in conjunction with a DNA polymerase possessing primer activation function, the method further includes: constructing a library and sequencing the linear amplification products. The library construction method is a technique well-known in the art and may include steps such as adapter ligation, pre-amplification, and library expansion; specific methods will not be elaborated here. The sequencing results are then analyzed to achieve rapid, sensitive, and accurate identification of pathogens and drug resistance genes in bloodstream infection samples.

[0067] The research process of the present invention will now be described in detail with reference to specific embodiments and accompanying drawings.

[0068] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in this invention all employ conventional techniques in molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related fields. Unless otherwise specified, all materials and reagents used in this invention are commercially available. Table 1 shows the materials and equipment used in this invention.

[0069] Table 1. Materials and Equipment Used in this Invention

[0070]

[0071] I. Design and Screening of Specific Single Primers

[0072] This invention involves the sequence design and screening optimization of specific single primers for 15 clinically important bloodstream infection pathogens (Candida albicans, Proteus mirabilis, Pseudomonas aeruginosa, Enterobacter cloacae, Serratia marcescens, Staphylococcus aureus, Enterococcus faecalis, Streptococcus pyogenes, Candida glabrata, Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae, Staphylococcus epidermidis, Streptococcus pneumoniae, and Enterococcus faecalis) and 4 drug resistance genes (mecA, OXA-48, NDM, and KPC).

[0073] (I) Sequence design of specific single primers

[0074] Each specific single primer contains a universal sequencing sequence and a specific sequence from the 5' end to the 3' end. The universal sequencing sequence is: 5'-GGCAGCGTCAGATGTGTATAAGAGACAG-3', and the specific sequence is a sequence that is complementary to the target region.

[0075] The sequence design method is as follows: Specific gene regions of 15 pathogens were extracted by alignment, and an initial primer set was designed for these regions. Each primer was screened under multiple conditions, including: (1) the length of the specific sequence was 20–40 bp; (2) the distance between the 3' end of the primer and the 5' end of the target region was as close as possible, within the range of 5–55 bp; (3) the specificity of the primers was evaluated using the Blast tool; (4) the annealing temperature of the primers was 65–80℃; (5) in Na… + Concentration of 100mM Mg 2+ Under the conditions of 0 mM and primer concentration of 100 nM, the melting temperature of each primer should be >64℃; (6) Analyze the homodimer and heterodimer probabilities of all primers and avoid using primers with strong ΔG (<-9 kcal / mol). Further, align all primers with the human genome, remove primer sequences with high homology to the human genome, and obtain a candidate primer set after screening.

[0076] Each pathogen and drug resistance gene candidate primer set contains 12 candidate primers, each named individually, totaling 228 primers. Primer names and target genes are shown in Tables 9 and 10 below. Each primer has a thiolated modification at the 3' end of the last three nucleotides of its phospholipid bond, and the -OH group of the 3' terminal nucleotide in all primers is replaced by a C6 spacer group. Primer synthesis was performed by Shanghai Yiming Biotechnology Co., Ltd.

[0077] (II) Primer screening and optimization

[0078] The OPERA targeted library preparation technology was used to detect pathogen reference materials. The detection sensitivity of each candidate primer at different working concentrations was evaluated, and the top three candidate primers with the highest detection sensitivity were selected as the final specific single primers and their corresponding working concentrations used in this application. The experimental methods and results are as follows:

[0079] I. Preparation of Pathogen Reference Materials

[0080] use The Genome DNA Extraction Kit (Cat#: APG-61002, Shanghai Yiming Biotechnology) extracts genomic DNA from Candida albicans, Proteus mirabilis, Pseudomonas aeruginosa, Enterobacter cloacae, Serratia marcescens, Staphylococcus aureus (resistance gene mecA), Enterococcus faecalis, Streptococcus pyogenes, Candida glabrata, Acinetobacter baumannii (resistance gene OXA-48), Escherichia coli (resistance gene NDM), Klebsiella pneumoniae (resistance gene KPC), Staphylococcus epidermidis, Streptococcus pneumoniae, and Enterococcus faecalis. The gDNA is fragmented by sonication for 500 seconds to obtain fragmented quality control samples. The main peak of pathogen DNA fragmentation is between 50bp and 100bp.

[0081] II. Use OPERA targeted library preparation technology to detect pathogen reference materials and evaluate the detection sensitivity of candidate primers.

[0082] Different pathogen reference samples were detected using 5× mixed primer working solutions and OPERA targeted library preparation reagents at working concentrations of 30 nM, 60 nM, 100 nM and 150 nM (see Table 1). The sample loading amount of the pathogen reference samples was 1000 copies.

[0083] The single-primer amplification-based targeted library construction kits were all provided by Shanghai Yiming Biotechnology Co., Ltd. The entire targeted process includes the following three steps: (1) linear amplification and affinity purification to obtain specific linear amplification products; (2) ligation reaction to obtain single-stranded library molecules; and (3) purification of the target double-stranded library molecules via Index PCR and library fragment purification. The specific steps are as follows:

[0084] 1. Single-primer linear amplification reaction

[0085] Refer to OPERA Follow the instructions in the Linear Amplification Kit (APG-62015-048) to perform the relevant operations.

[0086] Table 2 Preparation of linear amplification system

[0087] Components Volume (μL) 5×APO-Enchanted Buffer I 6 Bio-dNTP Mix 1 1.2 Apo-Enchanted DNA Polymerase I 0.2 5× Primer Working Solution 6 Pathogen reference material (1000 copies / μL) 1 water Supplement to 30 Total 30

[0088] APO-Enchanted DNA polymerase I is a high-fidelity DNA polymerase that can cleave the C6 spacer group using its 3'-5' exonuclease activity and incorporate biotinylated dNTPs.

[0089] (1) Prepare a Mix solution by mixing the components 5×APO-Enchanted Buffer I, Bio-dNTP Mix and Apo-Enchanted DNA Polymerase I in the kit according to Table 1.

[0090] (2) Add 5× primer working solution and sample (i.e. pathogen reference), and add water to make the reaction volume of each sample 30 μL.

[0091] (3) Start the linear amplification program: 98℃ pre-deformation for 100 seconds, 1 cycle; 95℃ denaturation for 10 seconds, 68℃ annealing and extension for 100 seconds, 50 cycles; 4℃ temporary storage.

[0092] 2. Purification of linear amplification products

[0093] Follow the instructions for use of the nucleic acid purification reagent (DPG-61101-048) to perform the relevant operations.

[0094] (1) Preparation of SA magnetic beads (add 20 μL of magnetic beads to each sample)

[0095] Remove the SA magnetic beads from the 4°C freezer and vortex for 30 seconds; add 1 mL of SA washing buffer 1 and mix by pipetting; place on a 1.5 mL magnetic rack and let stand for 2 minutes, discarding the supernatant; wash the magnetic beads once with 1 mL of SA washing buffer 1; resuspend in an appropriate volume of SA binding buffer. Add 0.25 μL of blocking reagent to each sample, mix well, and aliquot into 8-tube strips; block the magnetic beads at room temperature and 500-600 rpm for 10 minutes.

[0096] (2) Purification of SA magnetic beads

[0097] Add 20 μL of blocked magnetic beads to an 8-tube containing the linear amplification product; shake at 2000 rpm for 10-15 seconds until the magnetic beads are completely dispersed; shake at room temperature (500-600 rpm) for 15 minutes, then place on a 96-well magnetic rack and let stand for 2 minutes, discarding the supernatant; add 150 μL of SA washing buffer 1 and wash the magnetic beads 3 times; add 150 μL of SA washing buffer 2 and wash the magnetic beads 2 times; add 20 μL of SA elution buffer and shake at 3500 rpm for 2-5 minutes until the magnetic beads are completely dispersed; elute the target product at 80℃ for 4 minutes; after heating, let stand on a magnetic rack for 2-5 minutes, then transfer the supernatant to a new 8-tube for subsequent experiments.

[0098] 3. Connection reaction

[0099] Refer to the ssDNA single-strand ligation kit (APG-62002-096) and The connection products were obtained by following the instructions for using the single-chain tag short connector (APG-23026-120) for library construction. The connection system configuration is shown in Table 3, and the connection procedure is shown in Table 4.

[0100] Table 3. Connection System Configuration

[0101]

[0102] Table 4 Connection Program

[0103] Cycle number Temperature (°C) Time (minutes) 1 (Heat cap 105℃) 60 60 1 (Heat cap 105℃) 90 3 1 8 /

[0104] 4. Library pre-amplification reaction

[0105] Reference Library amplification reagents (for (APG-62003-096) Follow the instructions in the manual for using the pre-expansion tag primers (Set 1) (APG-23001-120) and nucleic acid purification reagent (DPG-61102-048) for library construction.

[0106] Table 5. Preparation of the pre-amplification reaction system

[0107]

[0108]

[0109] (1) Prepare the pre-amplification reaction system as shown in Table 5. The tag primer is added separately. According to the number of samples to be amplified, prepare the corresponding reaction system mix except for the pre-primer (Index) in a 1.5 mL centrifuge tube, and then dispense it into 8-tube strips. According to the library construction sample information table, add 1 μL of the corresponding tag primer to the corresponding reaction well.

[0110] (2) Add 24 μL of ligation product to the corresponding reaction well, and then place it in a PCR instrument for reaction. The reaction conditions are shown in Table 6 to obtain the pre-amplified product.

[0111] Table 6 Pre-amplification reaction program

[0112]

[0113] (3) Purify the pre-amplification product

[0114] Add an equal volume (50 μL) of NA binding buffer to the pre-amplified product; add 70 μL of NA magnetic beads. Vortex to mix the beads and incubate at 25°C for 5 minutes. Use a magnetic rack to hold the beads for 5 minutes until the solution is clear. Carefully aspirate the supernatant. Add 200 μL of 80% ethanol and wash twice. Finally, carefully remove the supernatant, avoiding aspirating the magnetic beads. After incubating at room temperature for 3 minutes, add 20 μL of NA elution buffer to fully suspend the beads and incubate at room temperature for 5 minutes to elute the DNA. Use a magnet to hold the beads and transfer the supernatant DNA solution to a new 8-tube strip. The product can be used directly for subsequent experiments.

[0115] 5. Library expansion

[0116] Reference Library amplification reagents (for Follow the instructions for use of (APG-62003-096) and nucleic acid purification reagent (DPG-61102-048) to perform relevant operations.

[0117] Table 7 Preparation of the expansion reaction system

[0118]

[0119]

[0120] (1) Prepare the corresponding reaction system according to Table 7 and dispense it into 8-tube bundles.

[0121] (2) Add the corresponding pre-amplified and purified product to the tube, centrifuge quickly for 10 seconds, and then place it in a PCR instrument to react and obtain the library amplification product. The library amplification reaction procedure is shown in Table 8.

[0122] Table 8. Expansion Response Procedure

[0123]

[0124] (3) Purification of NA magnetic beads after library expansion

[0125] Add 27 μL of NA magnetic beads; vortex to mix the beads thoroughly, then let stand at room temperature for 5 minutes; use a magnetic rack to adsorb the beads for 5 minutes until the solution is clear; carefully aspirate the supernatant; add 200 μL of 80% ethanol and wash twice; finally, carefully remove the supernatant, avoiding aspirating the magnetic beads; after standing at room temperature for 3 minutes, add 20 μL of NA elution buffer to fully suspend the beads, and let stand at room temperature for 5 minutes to elute the DNA; adsorb the beads with a magnet, and transfer the supernatant DNA solution to a new 8-tube; the product can be used directly for subsequent experiments. Take 2 μL of the purified product and determine the concentration using the Qubit quantitative reagent according to the instructions.

[0126] 6. Mixed Library

[0127] Based on the Qubit detection results, all libraries were mixed into 1.5 mL centrifuge tubes, with a sample volume of [volume missing] for each library. Where: V is the volume of the library to be mixed; X is the planned mass of the library to be mixed (ng, usually 5 ng); d is the mass concentration of the library to be mixed (ng / μL).

[0128] 7. Document Quality Control

[0129] The concentration of the library was determined using the Qubit dsDNA HS Assay Kit and its accompanying instruments. If the library concentration was lower than that required for sequencing, it could be concentrated or the library could be reconstructed.

[0130] The library was fragmented using the Agilent High Sensitivity DNA Kit and the accompanying Agilent 2200 Bioanalyzer system. The main peak of the fragment length should be between 170 bp and 300 bp, with no obvious small or large fragment peaks. If the fragment distribution of the library does not meet the requirements, the library should be reconstructed.

[0131] 8. Sequencing

[0132] This sequencing was performed using the Novaseq PE150 sequencing reagent / chip on an Illumina Novaseq 6000 sequencer, with approximately 2M of data allocated to each library.

[0133] 9. Data Analysis

[0134] The sequencing data was compared with the reference genome of the pathogen, the number of reads of the reference pathogen was counted, and the number of specific reads detected per megabyte of sequencing data was calculated by standardizing the sequencing data volume.

[0135] The experimental results are analyzed as follows:

[0136] (1) Screening results of candidate primers for pathogens

[0137] The results of the candidate primer screening for pathogens are shown in Table 9. The number of specific reads / M (the number of specific reads detected per M data) of each primer working solution was compared. The three primers with the highest values ​​were selected from the 12 primers (Y was selected in Table 9), and the primer concentration with the highest value was selected for subsequent primer working solution preparation.

[0138] Analysis of the data in Table 9 shows that the detection ability of the screened primers is 118%-259% of that before selection, with an average improvement of 179%. Meanwhile, the detection ability of the concentration-screened primers is 117%-144% of that before selection, with an average improvement of 125%.

[0139] Table 9. Results of candidate primer screening for pathogens

[0140]

[0141]

[0142]

[0143]

[0144]

[0145]

[0146] (2) Results of candidate primer screening for drug resistance genes

[0147] The results of the candidate primer screening for drug resistance genes are shown in Table 10. The number of specific reads / M (the number of specific reads detected per M data) of each primer working solution was compared. The three primers with the highest values ​​were selected from the 12 primers (Y was selected in Table 10), and the primer concentration with the highest value was selected for subsequent primer working solution preparation.

[0148] Analysis of the data in Table 10 shows that the detection ability of the screened primers is 115%-126% of that before selection, with an average improvement of 120%. Meanwhile, the detection ability of the concentration-screened primers is 123%-176% of that before selection, with an average improvement of 143%.

[0149] Table 10. Results of candidate primer screening for drug resistance genes.

[0150]

[0151]

[0152] Based on the above screening results, the specific sequences of the 19 sets of specific single primers finally adopted in this application are shown in Table 11, and the optimal working concentration for each primer is shown in Table 13 below.

[0153] Table 11 Primers for detecting cell-free nucleic acids of bloodstream pathogens

[0154]

[0155]

[0156]

[0157]

[0158]

[0159] (III) Detection of multiple pathogen reference materials using the specific single primers of this invention based on OPERA targeted library construction technology

[0160] The multiple pathogen reference samples included three types: Gram-positive bacteria, Gram-negative bacteria, and fungi. They were prepared separately in two groups, A and B, according to Table 12. The reference samples used ultrasonically fragmented pathogen DNA, with the main peak of the fragmented pathogen DNA between 50bp and 100bp. All reference samples contained fragmented human genomic DNA, categorized into high-incorporation (50ng) and low-incorporation (10ng) groups. Six gradient tests were set up for the reference samples: 3, 10, 30, 100, 400, and 1000 copies. Each test was repeated 4-6 times. The single primers and detection concentrations used were obtained from the screening in Experiment (II) above, as shown in Table 13. The experimental procedures were performed according to the relevant operations in Experiment (II) above.

[0161] Table 12 Grouping of Multiple Pathogen Reference Materials

[0162] Pathogen Name Species (Latin name) Drug resistance genes Classification Grouping Candida albicans Candida albicans Fungi A Proteus mirabilis Proteus mirabilis G- A Pseudomonas aeruginosa Pseudomonas aeruginosa G- A Enterobacter cloacae Enterobacter cloacae G- A Serratia marcescens Serratia marcescens G- A Staphylococcus aureus Staphylococcus aureus mecA G+ A Enterococcus faecalis Enterococcus faecium G+ A Streptococcus suis streptococcus mitis G+ A Candida glabrata Candida glabrata Fungi B Acinetobacter baumannii Acinetobacter baumannii OXA-48 G- B Escherichia coli Escherichia coli NDM G- B Klebsiella pneumoniae Klebsiella pneumoniae KPC G- B Staphylococcus epidermidis Staphylococcus epidermidis G+ B Streptococcus pneumoniae Streptococcus pneumoniae G+ B Enterococcus faecalis Enterococcus faecalis G+ B

[0163] Table 13. Single primers and detection concentrations used in the detection.

[0164]

[0165]

[0166] The test results are shown in Tables 14-15 and Figure 1A-1H As shown in the analysis results, the present invention greatly reduces the interference of human background cfDNA through targeted enrichment. The detection sensitivity of pathogens using the specific single primer of the present invention with OPERA technology is between 3 and 30 copies, which has extremely high detection sensitivity.

[0167] Table 14. Sensitivity and specificity analysis results of Group A reference samples

[0168]

[0169] Table 15 shows the sensitivity and specificity analysis results of the reference samples in Group B.

[0170]

[0171] (iv) Detection results of clinical samples from bloodstream infections

[0172] I. Patient and Sample Related Information

[0173] The inclusion criteria for patients with bloodstream infection are as follows: patients must meet any two of the following conditions 1 and 2: (1), (2), (3), (4), and (5) to be considered as having bloodstream infection. 1. Age not less than 18 years old; 2. Suspected bloodstream infection during hospitalization and requesting blood culture examination: (1) Meets the etiological diagnostic criteria for bloodstream infection: positive blood culture results or pathogens found in blood smears since the onset of the disease; (2) Elevated peripheral blood leukocyte count or increased neutrophil ratio that cannot be explained by non-infectious factors; (3) Elevated CRP / ESR / PCT / ferritin / PCT that cannot be explained by non-infectious factors; (4) Has at least one risk factor for bloodstream infection: recent history of invasive procedures, indwelling endovascular catheter before the onset of the disease, severe focal infection, echocardiography suggesting infective endocarditis, etc.; (5) Meets the diagnostic criteria for fever of unknown origin.

[0174] Patients are excluded if any of the following conditions are met: 1. Patients with incomplete clinical data; 2. Remaining blood samples are unusable due to insufficient volume, hemolysis, contamination, etc.; 3. Patients whose blood culture results are contaminated.

[0175] The patient had 2 mL of peripheral blood collected using an EDTA anticoagulant tube, centrifuged at 1000g for 5 minutes, and the plasma was separated.

[0176] II. Reagents and Equipment

[0177] Cell-free nucleic acids were extracted from plasma using nucleic acid extraction reagents (magnetic bead method) (catalog number DPG-61002) from Yiming (Shanghai) Diagnostics Co., Ltd. All other reagents and equipment were the same as in Example 1.

[0178] III. Experimental Procedure: Plasma extraction was performed according to the instructions for use of the nucleic acid extraction reagent (magnetic bead method) (catalog number DPG-61002) from Yiming (Shanghai) Diagnostics Co., Ltd. 1 mL of plasma was extracted from each sample.

[0179] Subsequent library construction and sequencing were performed in the same manner as in Experiment (II).

[0180] IV. Results Analysis

[0181] Tables 16-18 below show the statistics of infected samples, blood culture test results of enrolled patients, and OPERA technology test results, respectively. The analysis results show that, compared with conventional blood culture testing, this invention, based on OPERA technology and using the specific single primer provided by this invention to detect clinical samples of bloodstream infections, can significantly improve the positive rate of blood samples from patients with bloodstream infections, increasing it from 10.1% for blood culture to 58.9%. For blood culture-positive samples, OPERA testing also showed good consistency, reaching 88.2%.

[0182] Table 16 Statistics of Infected Samples

[0183] Infection type Sample count Bloodstream infections and cardiovascular infections 72 Gastrointestinal infection and intraperitoneal infection 41 Respiratory infection 25 Urinary tract infection 11 Skin and soft tissue infections 9 Central nervous system infection 7 Infectious fever or shock (source of infection unknown) 25 total 190

[0184] Table 17 Blood culture test results of enrolled patients

[0185]

[0186] Table 18. Results of OPERA technology testing (compared to blood culture)

[0187]

[0188] In summary, this invention aims to address the shortcomings of existing bloodstream infection detection methods in terms of speed, sensitivity, specificity, coverage, and cost through an innovative targeted linear amplification technology based on a dual-modified specific single primer and activated polymerase. It provides a faster, more sensitive, accurate, and cost-effective new solution for detecting multiple pathogens and drug resistance genes.

[0189] Although the present invention has been described in detail through the preferred embodiments above, it should be understood that the above description should not be considered as a limitation of the present invention. Various modifications and substitutions to the present invention will be apparent to those skilled in the art after reading the above description. Therefore, the scope of protection of the present invention should be defined by the appended claims.

Claims

1. A primer set for detecting cell-free nucleic acids of bloodstream pathogens, characterized in that, The primer set comprises the following 19 sets of primers: (1) Primers for detecting Acinetobacter baumannii free nucleic acid, the sequences of which are any one or more of SEQ ID NO: 1 to SEQ ID NO: 3; (2) Primers for detecting free nucleic acid of Candida albicans, the sequences of which are any one or more of SEQ ID NO: 4 to SEQ ID NO: 6; (3) Primers for detecting free nucleic acid of Candida glabrata, the sequences of which are any one or more of SEQ ID NO: 7 to SEQ ID NO: 9; (4) Primers for detecting free nucleic acid of Enterobacter cloacae, the sequences of which are any one or more of SEQ ID NO: 10 to SEQ ID NO: 12; (5) Primers for detecting Enterococcus faecalis free nucleic acid, the sequences of which are any one or more of SEQ ID NO: 13 to SEQ ID NO: 15; (6) Primers for detecting Enterococcus faecalis free nucleic acid, the sequences of which are any one or more of SEQ ID NO: 16 to SEQ ID NO: 18; (7) Primers for detecting free nucleic acid of Escherichia coli, the sequences of which are any one or more of SEQ ID NO: 19 to SEQ ID NO: 21; (8) Primers for detecting Klebsiella pneumoniae free nucleic acid, the sequences of which are any one or more of SEQ ID NO: 22 to SEQ ID NO: 24; (9) Primers for detecting free nucleic acid of Proteus mirabilis, having the sequence of any one or more of SEQ ID NO: 25 to SEQ ID NO: 27; (10) Primers for detecting free nucleic acid of Pseudomonas aeruginosa, the sequences of which are any one or more of SEQ ID NO: 28 to SEQ ID NO: 30; (11) Primers for detecting Serratia marcescens free nucleic acid, the sequences of which are any one or more of SEQ ID NO: 31 to SEQ ID NO: 33; (12) Primers for detecting free nucleic acid of Staphylococcus aureus, the sequences of which are any one or more of SEQ ID NO: 34 to SEQ ID NO: 36; (13) Primers for detecting free nucleic acid of Staphylococcus epidermidis, having the sequence of any one or more of SEQ ID NO: 37 to SEQ ID NO: 39; (14) Primers for detecting free nucleic acid of Streptococcus pyogenes, having the sequence of any one or more of SEQ ID NO: 40 to SEQ ID NO: 42; (15) Primers for detecting free nucleic acid of Streptococcus pneumoniae, the sequences of which are any one or more of SEQ ID NO: 43 to SEQ ID NO:

45. (16) Primers for detecting the drug resistance gene KPC, the sequences of which are any one or more of SEQ ID NO: 46 to SEQ ID NO: 48; (17) Primers for detecting the drug resistance gene mecA, the sequences of which are any one or more of SEQ ID NO: 49 to SEQ ID NO: 51; (18) Primers for detecting the drug resistance gene NDM, the sequences of which are any one or more of SEQ ID NO: 52 to SEQ ID NO: 54; (19) Primers for detecting the drug resistance gene OXA-48, the sequences of which are any one or more of SEQ ID NO: 55 to SEQ ID NO:

57.

2. The primer set as described in claim 1, characterized in that, Each primer has the following dual modifications: (1) Thio modification: the phospholipid bonds of the 3' end nucleotide backbone of the primer are thio modified; (2) Blocking modification: the 3' end of the primer has a blocking modification to prevent direct extension.

3. The primer set as described in claim 2, characterized in that, The thiomodification refers to the conversion of the phospholipid bonds in the nucleotide backbone from double bonds O to double bonds S.

4. The primer set as described in claim 2, characterized in that, The blocking modification is that the hydroxyl group at the 3-position of the nucleotide at the 3' end of the primer is replaced by a dimorphic functional group.

5. The primer set as described in claim 4, characterized in that, The dimorphic functional groups include any one or more of the following: phosphate groups, biotin groups, C6 spacer groups, and NH2-C6 groups.

6. A kit for detecting cell-free nucleic acids of bloodstream infection pathogens, characterized in that, The kit comprises the primer set according to any one of claims 1-5.

7. The kit according to claim 6, characterized in that, The kit also includes at least one of the following: plasma nucleic acid cell-free DNA extraction reagent, linear amplification reagent, adapter ligation reagent, or library construction reagent.

8. The kit according to claim 7, characterized in that, The linear amplification reagent contains DNA polymerase, dNTPs, and PCR amplification buffer.

9. The reagent kit as described in claim 8, characterized in that, The DNA polymerase has 3'-5' exonuclease activity.

10. The application of the kit according to any one of claims 6-9 in the detection of free nucleic acids of bloodstream pathogens.

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