A rapid bacterial identification technology based on 16S rRNA amplification-nanopore third-generation sequencing

Abstract

This invention belongs to the field of microbial pathogen identification technology and relates to a rapid bacterial identification technology based on 16S rRNA amplification-nanopore third-generation sequencing. It is applicable to the screening, confirmation and tracing of bacterial infections in multiple fields such as clinical medicine and public health monitoring. It is especially suitable for the accurate identification of bacterial pathogens that are difficult to isolate and culture using traditional culture methods, and provides technical support for the rapid diagnosis, prevention and control and treatment guidance of infectious diseases.

Description

Technical Field

[0001] This invention belongs to the field of microbial pathogen identification technology, specifically involving a rapid bacterial identification technology based on 16S rRNA amplification-nanopore third-generation sequencing. Background Technology

[0002] Infectious diseases are a major global public health problem, with bacterial infections being one of the most common types of infections in clinical practice. These infections encompass a variety of conditions, including sepsis, pneumonia, tuberculosis, urinary tract infections, and abdominal infections, seriously threatening human life and health. Rapid and accurate identification of causative pathogens is a core prerequisite for effective prevention and targeted treatment of infectious diseases, and thus has a strong and urgent clinical need.

[0003] Sepsis is a life-threatening disease that occurs when the body's immune system reacts extremely to an infection, leading to organ dysfunction, and is usually caused by bacterial infection. According to data released in 2020, there were 48.9 million cases of sepsis and 11 million sepsis-related deaths worldwide, accounting for 20% of all deaths globally. The overall case fatality rate for sepsis patients is approximately 26.7%, and the case fatality rate for sepsis patients in ICUs is 41.9%. Furthermore, for patients with septic shock, survival rates decrease with every hour of delay in receiving targeted treatment; therefore, rapid identification of the bacterial pathogen is crucial for saving lives and improving prognosis. Simultaneously, with the irrational use of broad-spectrum antibiotics in clinical practice, bacterial resistance continues to rise globally. In 2019, an estimated 4.95 million deaths were related to antimicrobial resistance, of which 1.27 million were directly attributable to antimicrobial resistance.

[0004] The need to further enhance the urgency and accuracy of bacterial pathogen identification is urgent. Efficient identification technologies are needed to provide a basis for targeted drug use, reduce the overuse of broad-spectrum antibiotics, curb the development of drug resistance, and reduce the difficulty of treating infectious diseases and mortality rates. Summary of the Invention

[0005] This invention provides a rapid identification method for bacterial pathogens based on 16S rRNA gene amplification nanopore third-generation sequencing. It is applicable to the screening, confirmation and tracing of bacterial infections in multiple fields such as clinical medicine and public health monitoring. It is especially suitable for the accurate identification of bacterial pathogens that are difficult to isolate and culture using traditional culture methods, and provides technical support for the rapid diagnosis, prevention and control and treatment guidance of infectious diseases.

[0006] In a first aspect of the invention, an apparatus for detecting bacterial infection in a sample is provided, the apparatus comprising the following modules: (z1) Sample providing module, the sample providing module being configured to provide a sample to be tested; (z2) Amplification module, the amplification module being configured to amplify the sample to be tested using specific primer pairs, thereby obtaining amplification products; (z3) Sequencing module, the sequencing module being configured to: construct a sequencing library based on the amplification product and perform sequencing, thereby providing sequencing results; (z4) Result output module, the result output module being configured to: provide the bacterial infection status of the sample to be tested based on the sequencing results; The specific primer pair comprises an upstream primer and a downstream primer, wherein the upstream primer comprises the sequence shown in SEQ ID NO:1 and the downstream primer comprises the sequence shown in SEQ ID NO:2.

[0007] In another preferred embodiment, the sample providing module is configured to provide sample nucleic acid derived from the sample to be tested.

[0008] In a specific implementation, the sample providing module is configured to: provide a sample to be tested, extract total nucleic acid from the sample to be tested, thereby providing sample nucleic acid.

[0009] In another preferred embodiment, the amplification module is configured to amplify the nucleic acid of the sample using specific primer pairs to obtain amplification products.

[0010] In a specific implementation, the amplification module is configured to provide specific primer pairs and amplification-essential reagents to amplify the sample to be tested, thereby obtaining amplification products.

[0011] In another preferred embodiment, the amplification-essential reagent refers to an amplification reagent used in a one-step PCR amplification reaction.

[0012] In another preferred embodiment, the amplification-essential reagents include: hot-start DNA polymerase, reverse transcriptase, dNTPs, magnesium ions, buffer solution, and enzyme-free water.

[0013] In another preferred embodiment, the amplification-essential reagent comprises an inert reference dye.

[0014] In another preferred embodiment, the inert reference dye comprises: Rhodamine X (ROX).

[0015] In another preferred embodiment, the sample to be tested is a sample without bacterial infection, a sample with one type of bacterial infection, or a sample with multiple types of bacterial infection.

[0016] In another preferred embodiment, "multiple" means ≥10 kinds, more preferably ≥12 kinds, even more preferably ≥15 kinds, even more preferably ≥18 kinds, and most preferably ≥20 kinds.

[0017] In another preferred embodiment, the species of bacteria infecting the sample to be tested is completely unknown.

[0018] In a specific implementation, the test sample is a human sample.

[0019] In another preferred embodiment, the test sample is a test sample derived from a mammal.

[0020] In another preferred embodiment, the mammals include: mice, rats, rabbits, dogs, cattle, sheep, camels, pigs, and non-human primates.

[0021] In another preferred embodiment, the test sample is a sample derived from a chicken or duck.

[0022] In a specific implementation, the sample to be tested includes: blood, plasma, bronchoalveolar lavage fluid, cerebrospinal fluid, pharyngeal swab, sputum, urine, or breast milk.

[0023] In another preferred embodiment, the bacterial infection status of the sample includes: (i) Whether there is bacterial infection in the sample; (ii) The species of bacteria infecting the sample; (iii) The relative abundance ratio of each infecting bacterial species in the sample.

[0024] In another preferred embodiment, the sequencing includes: second-generation sequencing and third-generation sequencing.

[0025] In another preferred embodiment, the upstream primer is shown in SEQ ID NO:7 and the downstream primer is shown in SEQ ID NO:8; The sequencing in question refers to nanopore sequencing.

[0026] In another preferred embodiment, the sequencing module is configured to provide a nanopore sequencing component to construct a sequencing library and perform nanopore sequencing on the amplified products, thereby providing sequencing results.

[0027] In another preferred embodiment, the nanopore sequencing assembly comprises: a sequencer, a sequencing chip, a Qubit quantitative analyzer, or a combination thereof.

[0028] In another preferred embodiment, the nanopore sequencing component includes a computer server.

[0029] In another preferred embodiment, the device is a device for broadly screening bacterial infection samples.

[0030] In another preferred embodiment, the detection covers ≥10 bacterial species, more preferably ≥12 species, even more preferably ≥15 species, further preferably ≥18 species, and most preferably ≥20 species.

[0031] In another preferred embodiment, the bacteria comprise: Gram-positive bacteria, Gram-negative bacteria, or a combination thereof.

[0032] In another preferred embodiment, the bacteria comprise: Mycobacterium tuberculosis, Streptococcus pneumoniae, Moraxella catarrhalis, Haemophilus influenzae, Legionella pneumophila, Klebsiella pneumoniae, Staphylococcus aureus, Group A beta-hemolytic streptococci, Pseudomonas aeruginosa, Acinetobacter baumannii, Escherichia coli, Neisseria meningitidis, Neisseria microflagellate, Corynebacterium stripe, or combinations thereof.

[0033] In another preferred embodiment, the amplification reaction is a one-step amplification reaction.

[0034] In another preferred embodiment, the amplification process includes: incubation at 42°C for 30 min; pre-denaturation at 95°C for 10 sec; (denaturation at 95°C for 15 sec, annealing at 50°C for 60 sec, extension at 72°C for 90 sec) for 30 cycles; final extension at 72°C for 5 min; and storage at 4°C.

[0035] In another preferred embodiment, the construction of the sequencing library includes: extracting nucleic acids from the sample to be tested, and performing full-length 16S rRNA amplification using a one-step method; purifying the amplification product, merging multiple samples in equal amounts, ligating Nanopore sequencing adapters, and constructing a sequencing library; performing Nanopore sequencing, and performing bioinformatics analysis on the data to detect bacteria and identify species.

[0036] In a second aspect of the invention, a kit is provided, the kit comprising: (a) A primer pair comprising the sequence shown in SEQ ID NO:1 and the sequence shown in SEQ ID NO:2; (b) Amplification-essential reagents, wherein the amplification-essential reagents include: hot-start DNA polymerase, reverse transcriptase, dNTPs, magnesium ions, buffer, enzyme-free water, or combinations thereof.

[0037] In another preferred embodiment, the kit further comprises: (c) Nanopore sequencing reagents, wherein the nanopore sequencing reagents include: RLB sample tag sequences, RAP adapters, magnetic beads, or combinations thereof.

[0038] In a third aspect of the invention, the use of the kit described in the second aspect of the invention is provided for preparing a detection product for detecting bacterial infection in a sample, and / or for broad-spectrum screening of bacterially infected samples.

[0039] In a fourth aspect of the invention, a method for detecting bacterial infection in a sample for non-diagnostic purposes is provided, comprising the following steps: (S1) Provide the sample to be tested; (S2) The sample to be tested is amplified using a specific primer pair to obtain the amplification product; (S3) Construct a sequencing library based on the amplification primers and perform sequencing to provide sequencing results; (S4) Based on the sequencing results, provide the bacterial infection status of the sample to be tested; The specific primer pair comprises an upstream primer and a downstream primer, wherein the upstream primer comprises the sequence shown in SEQ ID NO:1 and the downstream primer comprises the sequence shown in SEQ ID NO:2.

[0040] In another preferred embodiment, the upstream primer is shown in SEQ ID NO:7 and the downstream primer is shown in SEQ ID NO:8; The sequencing in question refers to nanopore sequencing.

[0041] In another preferred embodiment, the sample to be tested includes: a food sample, an environmental sample, or a combination thereof.

[0042] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0043] Figure 1 The flowchart shows the experimental procedure for rapid identification of bacterial pathogens using 16S rRNA gene amplification and third-generation nanopore sequencing.

[0044] Figure 2 Electrophoresis diagrams of primer amplification using 16S and RPB004-16S primers are shown. Detailed Implementation

[0045] Through extensive and in-depth research, and through numerous screenings and repeated experiments, the inventors unexpectedly discovered for the first time a specific set of degenerate primers targeting the full-length 16S rRNA long read of Nanopore. Using these specific degenerate primers, this invention provides a rapid bacterial identification method based on 16S rRNA amplification-nanopore third-generation sequencing. This method is characterized by high specificity, broad spectrum, high detection accuracy, and high sensitivity. Based on this method, this invention can perform preliminary screening of samples with completely unknown infection status, and preliminarily obtain identification results for various bacterial species. This invention was completed based on this foundation.

[0046] Specifically, the method of this invention addresses the core problems of traditional pathogen identification methods in the diagnosis and treatment of infectious diseases, such as long identification cycles, inability to identify pathogenic bacteria that are difficult to culture, and low accuracy. It can significantly shorten the identification cycle to within 36 hours, and in some emergency scenarios, preliminary results can be obtained within 12 hours. It can accurately identify unculturable bacterial pathogens, while simplifying the operation process and reducing testing costs. It meets the urgent needs of rapid diagnosis and treatment and precise medication for clinical infectious diseases, and provides a reliable technical means for the precise prevention and treatment of bacterial infectious diseases such as sepsis and pneumonia. It effectively reduces the abuse of broad-spectrum antibiotics, curbs the development of bacterial resistance, and reduces the mortality rate of infectious diseases. It has important clinical practical value and public health significance.

[0047] the term To facilitate a clearer understanding of this disclosure, certain terms are first defined. As used herein, unless otherwise expressly specified herein, each of the following terms shall have the meaning given below. Other definitions are set forth throughout the application.

[0048] As used herein, the term “and / or” refers to and covers any and all possible combinations of one or more of the related listed items.

[0049] As used herein, the terms “comprising,” “including,” and “containing” are used interchangeably and include not only closed definitions but also semi-closed and open definitions. In other words, the terms include “consisting of” and “substantially consisting of”.

[0050] As used in this article, the term "or a combination thereof" means "or any combination thereof".

[0051] As used in this article, the term "16S rRNA gene" refers to the gene encoding the 16S small subunit of the bacterial ribosome, which is highly conserved in bacteria and species-specific.

[0052] As used in this article, the term "RNase" refers to ribonuclease, a class of hydrolytic enzymes that can specifically cleave and degrade RNA (ribonucleic acid). These enzymes are widely found in biological tissues, cells, the environment, and routine laboratory consumables. They can decompose RNA in samples, causing nucleic acid degradation and template invalidation, severely interfering with molecular experiments such as RT-PCR, nucleic acid extraction, gene amplification, and sequencing library preparation.

[0053] As used in this article, the term "one-step RT-PCR" refers to a technique in which reverse transcription (RT) and PCR amplification are performed continuously in the same reaction system and the same procedure, without the need for step-by-step operations, simplifying the process and reducing contamination.

[0054] As used in this article, the term "library construction" refers to the standardized experimental procedure of ligating adapters and modifying molecules of purified PCR products to adapt them for sequencing chip detection.

[0055] As used in this article, the term "Nanopore sequencing" refers to third-generation single-molecule real-time sequencing technology, represented by the MiniON device. It features short sequencing cycles, real-time data analysis, and suitability for rapid clinical testing.

[0056] As used in this article, the term "whole genome sequencing" refers to sequencing the complete bacterial genome, which has extremely high identification accuracy, but is costly and involves complex data analysis.

[0057] As used in this article, the term "metagenomic sequencing" (mNGS) refers to high-throughput sequencing of all microbial nucleic acids in clinical samples without targeted amplification, enabling unbiased screening of bacteria, fungi, and unknown pathogens.

[0058] As used herein, the term "degenerate primer" refers to a set of oligonucleotides with distinct sequences designed for a specific gene family or homologous sequence, rather than a single sequence. In specific implementations, in addition to the conventional A (adenine), C (cytosine), G (guanine), and T (thymine), degenerate base symbols established by the International Union of Pure and Applied Chemistry (IUPAC) are introduced to represent multiple base combinations at specific positions. The specific rules are as follows: R represents A or G (purine), Y represents C or T (pyrimidine), S represents G or C, W represents A or T, K represents G or T, M represents A or C, D represents A, G, or T (i.e., all bases except C), V represents A, C, or G (except T), H represents A, C, or T (except G), B represents C, G, or T (except A), and N represents any one of the bases A, C, G, or T. In physical synthesis, these symbols represent the equimolar mixing of the corresponding nucleotide monomers at that site, which effectively reduces the complexity of the primer library while maximizing the possibility of annealing and binding with the target gene template.

[0059] As used herein, the term "inert reference dye" refers to a dye that is detectable by the PCR instrument during a PCR reaction. It does not participate in the PCR reaction. In specific implementations, many physical factors inherent to the qPCR reaction can affect signal detection, leading to variations in well data. Inert reference dyes can eliminate these errors through homogenization correction, thereby improving data accuracy.

[0060] As used in this article, the term "RLB sample tag sequence" refers to a specific tag sequence introduced during Nanopore sequencing library preparation that can be used to distinguish samples. By tagging different samples with different RLB tag sequences, multiple samples can be detected simultaneously in one sequencing run. Then, through subsequent sequencing data analysis, the sequences from different samples can be separated by the tag sequences.

[0061] As used in this article, the term "RAP adapter" refers to a nucleic acid-protein complex adapter pre-loaded with motor proteins, which is specific to the ONT Nanopore Rapid Transposition Library Preparation Kit. Motor proteins are the core functional component carried on the RAP adapter. The principle of ONT nanopore sequencing is that motor proteins attach to the ends of the DNA fragment to be tested via sequencing adapters. Under the pull of an electric field, the negatively charged DNA is slowly and uniformly pulled through the nanopore by the motor proteins, relying on the hydrolysis of ATP, thereby achieving base recognition of the DNA sequence.

[0062] Identification of bacterial pathogens Currently, the mainstream method for identifying bacterial pathogens in clinical practice is still the traditional clinical culture method. This method requires isolating and culturing bacteria from clinical samples (such as blood, bronchoalveolar lavage fluid, urine, cerebrospinal fluid, etc.) on artificial culture media. After visible colonies form, identification is completed through morphological observation and biochemical tests. However, this method has many insurmountable drawbacks, which seriously restrict the efficiency and effectiveness of diagnosis and treatment of infectious diseases and cannot meet the needs of precise and rapid clinical diagnosis and treatment. First, the long identification cycle severely delays the treatment of infectious diseases. Traditional culture methods typically require 48-72 hours to obtain preliminary identification results, and for some fastidious bacteria (such as Mycobacterium tuberculosis and Streptococcus pneumoniae, common pathogens), the culture time can even be as long as 5-30 days. However, in clinical practice, infectious diseases, especially acute and severe infections, often require pathogen identification results within 24 hours to formulate targeted treatment plans. This delay often prevents patients from receiving targeted therapy in a timely manner, forcing them to rely on empirical medication, which not only has poor treatment effects but also easily leads to the spread of infection and worsening of the condition. Delayed bacterial identification may increase the mortality rate of patients with severe infections.

[0063] Secondly, the inability to identify difficult-to-culture and unculturable pathogenic bacteria leads to missed or misdiagnosed infectious diseases. Clinical infection samples contain a large number of pathogenic bacteria that are difficult to isolate and culture using artificial culture media (i.e., difficult-to-culture bacteria). Some pathogenic bacteria cannot even grow under artificial conditions. For these unculturable bacterial infections (such as pneumonia and abdominal infections caused by some anaerobic or fastidious bacteria), traditional methods cannot identify the pathogen. This results in some clinical infection cases having no clear cause, requiring empirical treatment with broad-spectrum antibiotics. This not only has poor treatment efficacy but also increases the risk of bacterial resistance and may even lead to prolonged and recurrent infections.

[0064] Third, the limited accuracy and cumbersome procedures of traditional culture methods affect the precision of infectious disease diagnosis and treatment. Traditional culture methods rely on the experience and judgment of laboratory personnel, which can easily lead to misjudgments and thus deviations in treatment plans. Furthermore, the cumbersome procedures of traditional methods require manual intervention throughout the entire process of clinical sample processing, culture, and identification, increasing the risk of contamination and further raising the failure rate and cost of repeated testing, thus delaying the diagnosis and treatment of infectious diseases.

[0065] Fourth, existing molecular biology identification methods cannot meet the routine diagnostic and treatment needs of clinical infectious diseases. Although some molecular biology techniques (such as PCR) can shorten the identification time, they can only target known specific pathogens and cannot screen for unknown pathogens, making them unsuitable for infectious diseases with unknown causes in clinical practice. While whole-genome sequencing technology can achieve accurate identification, its high sequencing cost, complex data analysis, and long cycle make it difficult to widely apply to routine clinical testing scenarios, especially unsuitable for the rapid diagnosis and treatment needs of primary healthcare institutions and emergency infectious diseases.

[0066] 16S rRNA gene As a conserved gene in bacteria, the 16S rRNA gene has species-specific sequences. Amplification and sequencing technology based on the 16S rRNA gene can overcome the limitations of traditional culture methods. Without the need for bacterial isolation and culture, nucleic acids can be extracted directly from clinical samples of patients (blood, bronchoalveolar lavage fluid, urine, cerebrospinal fluid, etc.), and the 16S rRNA gene can be amplified and sequenced. Species identification of pathogens can be quickly completed through bioinformatics analysis.

[0067] The method of the present invention This invention provides a 16S amplification sequencing method for pathogen identification. Compared to existing pathogen identification technologies that rely primarily on traditional bacterial culture supplemented by conventional PCR and whole-genome sequencing, this invention, based on 16S rRNA gene amplification sequencing, fundamentally overcomes the shortcomings of existing technologies, such as long identification cycles, inability to detect difficult-to-culture bacteria, low accuracy, and limited applicability. Traditional culture methods require 48–72 hours to produce results, and for fastidious bacteria, even 5–30 days, which can delay the treatment of severe infections such as sepsis. Furthermore, they cannot identify difficult-to-culture or unculturable pathogens, leading to missed or misdiagnosed cases. They also rely on manual experience, are cumbersome, and carry a high risk of contamination. Conventional PCR can only target known pathogens and cannot screen for unknown pathogens, while whole-genome sequencing is difficult to implement routinely in clinical practice due to its high cost and complex analysis.

[0068] This invention, based on the principles of 16S rRNA gene conservation and species specificity, eliminates the need for bacterial isolation and culture. It allows for direct nucleic acid amplification, sequencing, and bioinformatics analysis from clinical samples such as blood, bronchoalveolar lavage fluid, and cerebrospinal fluid. On one hand, it significantly reduces the identification cycle to within 36 hours, and preliminary results can be obtained within 12 hours in emergency situations, solving the problems of excessively long processing times and missed treatment windows associated with existing technologies. On the other hand, it can accurately identify difficult-to-culture and unculturable pathogenic bacteria, avoiding missed diagnoses and misdiagnoses. It also eliminates reliance on manual experience, simplifies procedures, reduces contamination and testing costs, and enables screening for unknown pathogens, overcoming the limitations of PCR and whole-genome sequencing. This invention not only meets the needs for rapid and accurate diagnosis and treatment of infectious diseases, guiding targeted drug use, reducing the overuse of broad-spectrum antibiotics, and curbing bacterial resistance, but it is also applicable to routine and emergency testing in medical institutions at all levels, effectively reducing the mortality rate of severe infections and possessing significant clinical application and public health value.

[0069] The main advantages of this invention include: (a) The one-step clinical bacterial infection sample pathogen identification method based on 16S rRNA gene amplification combined with nanopore sequencing provided by the present invention integrates RPB004-16S primers and RPB004 barcodes into the same reaction system, completing 16S gene amplification and barcode labeling in one step. It eliminates the need for bacterial isolation and culture in clinical samples, and directly extracts nucleic acid from clinical samples to complete the complete process of one-step amplification, library construction, sequencing and pathogen identification in sequence. It eliminates the need for step-by-step experiments, simplifies the process, shortens the detection cycle to within 36 hours, and provides preliminary results within 12 hours in emergency scenarios.

[0070] (b) The specific primer combination provided by the present invention for the method of the present invention introduces a degenerate base design, which has good versatility and fault tolerance. It can be broadly adapted to the 16S rRNA gene template of different kinds of bacteria, and can achieve efficient and specific amplification of various clinical pathogens, difficult-to-culture bacteria and fastidious bacteria. It effectively improves the coverage and accuracy of pathogen detection and identification. It can be used to screen bacterial infection targets in the field of public health, and can also quickly determine whether a specific test subject has a bacterial infection.

[0071] (c) The method of this invention can not only detect whether a sample is infected with bacteria, but also detect the number of different types of bacterial infections present in the sample, and provide the relative abundance of each type of bacteria. Based on this, in the treatment of bacterial infections, targeted treatment can be administered according to the type and proportion of the infecting bacteria, achieving precision medicine.

[0072] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0073] Example 1: Experimental method.

[0074] like Figure 1 As shown, this invention uses samples (such as plasma, bronchoalveolar lavage fluid, cerebrospinal fluid, pharyngeal swabs, etc.) from patients with suspected bacterial infections as the detection targets, and employs the one-step 16S amplification combined with nanopore sequencing technology described in this invention for pathogen identification.

[0075] The experimental reagents used are as follows: Magnetic bead nucleic acid extraction kit (containing magnetic beads, lysis buffer, washing buffer, etc.), OneStep PrimeScript RT-PCR kit (catalog number RR064A, TAKARA, including 2×one-step RT-PCR buffer). Ex TaqHS, RT Enzyme, RNase-free dH2O, etc.), 16S rRNA amplification primers, SQK-RPB114 library preparation kit (ONT, including RLB sample tag sequence, RAP sequencing adapter, etc.), VAHTS DNA Clean Beads (Vazyme), 70% ethanol, elution buffer (10 mM Tris-HCl pH 8.0, 50 mM NaCl), Qubit quantitative kit Equalbit dsDNA HS Assay Kit (Vazyme, EQ111).

[0076] Collect specimens from patients with suspected bacterial infections, such as plasma, bronchoalveolar lavage fluid, pharyngeal swabs, and cerebrospinal fluid, place them in sterile centrifuge tubes, store them at 4°C, and send them for testing within 2 hours after collection.

[0077] Specifically, it includes the following steps: (1) Total nucleic acid extraction Total nucleic acid was extracted from the collected clinical samples according to the instructions of the magnetic bead-based nucleic acid extraction kit to obtain sample nucleic acid. A blank pure water control group was also established to assess and rule out contamination of the testing system.

[0078] (2) One-step amplification reaction For each batch of samples, positive and negative control samples were set up in parallel for amplification of 16S bacterial rRNA. The specific procedures are as follows: (2.1) Preparation of the reaction system: In a sterile operating room, prepare the amplification reaction system according to the proportions in the table below. First, prepare the mixture (excluding sample nucleic acid), then aliquot it into each reaction tube, and finally add the sample nucleic acid to avoid cross-contamination. The single-tube amplification reaction system (25 μL) is shown in Table 1.

[0079] Table 1 (2.2) Amplification system setup: Place the prepared amplification reaction tubes into the PCR instrument, set the reaction program as follows, and start the amplification: Step 1: Incubate at 42℃ for 30 minutes to complete the reverse transcription reaction; Step 2: Pre-denaturate at 95℃ for 10 seconds to activate the hot-start enzyme; Step 3: 30 cycles, each cycle consisting of 95°C denaturation for 15 seconds, 50°C annealing for 60 seconds, and 72°C extension for 90 seconds; Step 4: Final extension at 72℃ for 5 minutes to ensure the integrity of the amplification product; Step 5: After amplification, store the reaction tube at 4°C to prevent degradation of the amplification products.

[0080] (2.3) Purification of PCR products Add an equal volume (25 μL) of VAHTS DNA CleanBeads magnetic beads to 25 μL of PCR product in each amplification reaction tube, gently vortex to mix, avoiding vigorous shaking that could break the magnetic beads, and incubate at room temperature for 5 min to allow the amplification product to fully bind with the magnetic beads. Place the centrifuge tube on the magnetic rack and let it stand for 2 minutes until the magnetic beads are completely adsorbed. Then, slowly aspirate the supernatant, being careful not to remove the magnetic beads. Add 200 μL of 70% sterile ethanol (freshly prepared) to the centrifuge tube to wash the magnetic beads. Let it stand for 1 minute and then aspirate the supernatant. Repeat the ethanol washing step twice. Leave the magnetic beads on the magnetic rack and air dry at room temperature for 3-5 minutes until there is no ethanol residue on the surface of the magnetic beads (avoid over-drying, otherwise the nucleic acid will be difficult to elute); add 12 μL (10 mM Tris-HCl pH 8.0, 50 mM NaCl) to each centrifuge tube, mix by pipetting 10 times, and incubate at room temperature for 2 minutes to elute the amplification products from the magnetic beads. Place the centrifuge tube back on the magnetic rack and let it stand for 2 minutes until the magnetic beads are completely adsorbed. Then, carefully aspirate 11 μL of the supernatant (i.e., the purified PCR product) with a pipette and transfer it to a new sterile eight-tube set. Label the tube for subsequent quantification.

[0081] (2.4) Sample quantification and pooling Quantitative preparation: Prepare the standards and detection reaction solution according to the Equalbit dsDNA HS Assay Kit instructions; Qubit quantification: 1 μL of purified PCR product was added to the detection reaction solution, vortexed and mixed, and incubated at room temperature for 2 min. The Qubit detector was used for quantification, the concentration of each amplified product was recorded, and the molar number (fmol) of each product was calculated based on the length of the amplified product. Sample pooling: Based on the quantitative results, take an equal amount of each sample and pool it into the same 1.5 ml nuclease-free centrifuge tube to make the total amount of product after pooling reach 100 fmol; if the pooled volume is less than 10 μL, dilute it to a final volume of 10 μL with elution buffer (10 mM Tris-HCl pH 8.0, 50 mM NaCl), gently tap to mix, and then centrifuge briefly to avoid liquid residue on the tube wall.

[0082] (2.5) Document library connector connection Take 10 μL of the merged purified product, add 1 μL of RAP adapter, gently mix, and incubate at room temperature for 5 min. Ensure that the adapter is fully ligated to the DNA fragment to complete the construction of the nanopore sequencing library. After the library construction is completed, immediately store it on ice to avoid adapter degradation. Load the sample after the chip is ready.

[0083] (2.6) MiniON sequencing and data analysis Following the ONT MiniON sequencing chip sequencing sample loading protocol, the sequencing chip quality control, air bubble removal, and sample loading steps were completed before real-time sequencing was performed. Sequencing was terminated after 2 hours to allow for data analysis. Sequencing data processing: After sequencing, Emu, a species-level microbial community abundance analysis tool for Nanopore full-length 16S rRNA long reads, was used. The Expectation-Maximization (EM) algorithm was employed to correct sequencing errors and accurately estimate relative species abundance. This tool determines the species of pathogenic bacteria in clinical samples by globally comparing the full-length 16S long reads with reference databases (rrnDB + NCBI 16S RefSeq), thus completing pathogen identification. For difficult-to-culture or unculturable bacteria, sequence alignment enables accurate identification, avoiding missed diagnoses and misdiagnoses.

[0084] Example 2: Optimization of amplification primers.

[0085] In this embodiment, three sets of amplification primers were designed to target the full-length 16S rRNA (1.5 kb), the V3-V4 variable region (470 bp), and the V4 variable region (300 bp), respectively. The primer sequences are detailed below.

[0086] PCR amplification and agarose gel electrophoresis were performed using bronchoalveolar lavage fluid samples from patients with clinically positive bacterial infections and negative control samples. The results showed that all three primer groups could achieve effective amplification; among them, the full-length 16S rRNA primer amplified the brightest band and had the best amplification specificity. Figure 2 ).

[0087] 1) 16S primer pair Full-length primer (1.5kb) F1: AGRGTTYGATYMTGGCTCAG (SEQ ID NO:1) R1: RGYTACCTTGTTACGACTT (SEQ ID NO: 2) V3-4 variable region (470bp) F2: CCTAYGGGRBGCASCAG (SEQ ID NO:3) R2: GGACTACNNNGGGTATCTAAT (SEQ ID NO:4) V4 variable region (300bp) F3: GTGCCAGCMGCCGCGGTAA (SEQ ID NO:5) R3: GGACTACHVGGGTWTCTAAT (SEQ ID NO:6) 2) RPB004-16S primer pair Furthermore, to ensure 16S amplification compatibility with downstream Nanopore sequencing, the 16S primer sequence was ligated with the Nanopore sequencing adapter sequence to synthesize the RPB004-16S primer pair, which was then validated by PCR amplification and agarose gel electrophoresis. The results showed that all three primer sets could achieve effective amplification, with the RPB004-16S rRNA full-length primer pair exhibiting the highest band brightness and the best amplification specificity. Figure 2 Meanwhile, the RPB004-16S full-length amplified product fragment is the longest, and sequencing yields richer species differentiation information, resulting in higher accuracy in bacterial identification. Therefore, all subsequent amplification and sequencing analyses in this study used full-length primer pairs. The RPB004-16S primer pair sequences are as follows: Full-length primer (1.5kb): F1: CGTTTTTCGTGCGCCGCTTCAGRGTTYGATYMTGGCTCAG (SEQ ID NO:7) R2: CGTTTTTCGTGCGCCGCTTCRGYTACCTTGTTACGACTT (SEQ ID NO:8) V3-4 variable region (470bp): F2: CGTTTTTCGTGCGCCGCTTCCCTAYGGGRBGCASCAG (SEQ ID NO:9) R2: CGTTTTTCGTGCGCCGCTTCGGACTACNNNGGGTATCTAAT (SEQ ID NO: 10) V4 variable region (300bp): F3: CGTTTTTCGTGCGCCGCTTCGTGCCAGCMGCCGCGGTAA (SEQ ID NO: 11) R3: CGTTTTTCGTGCGCCGCTTCGGACTACHVGGGTWTCTAAT (SEQ ID NO: 12) It is noteworthy that the RPB004-16S rRNA full-length primers of this invention are degenerate primers designed after extensive screening and validation. Traditional 16S rRNA full-length primers amplify bands that are missing, while the degenerate primers provided in this application amplify bands with the highest brightness and the best amplification specificity.

[0088] Example 3: The method of the present invention is used for broad-spectrum detection of bacteria.

[0089] This embodiment evaluates the amplification suitability of 16S full-length degenerate primers for common bacteria causing clinical respiratory, central nervous system, and intestinal infections (see Table 2) by comparing their genome sequences.

[0090] Table 2 The comparison results indicate that the primer pair has excellent matching effect with the conserved 16S regions of all tested bacterial species, and can stably achieve target sequence binding of multiple pathogenic bacteria. This proves that the design of this set of full-length degenerate 16S primers is scientific and reliable, perfectly matches the bacterial sequence, and is suitable for broad-spectrum amplification detection of bacteria infected in the above-mentioned multiple sites.

[0091] Example 3: Sequencing of clinical specimens.

[0092] In this implementation case, two bronchoalveolar lavage fluid specimens identified by qPCR as respiratory pathogen infections were subjected to one-step 16S amplification combined with nanopore sequencing for pathogen identification. The sequencing results showed that the one-step 16S amplification sequencing method could successfully detect the corresponding pathogens. The number of bacterial-specific reads and relative abundance of species in 16S sequencing are shown in Table 3.

[0093] Table 3 This embodiment demonstrates that the one-step 16S amplification combined with nanopore sequencing pathogen identification method of the present invention is simple to operate, fast, efficient, and highly accurate. It does not require bacterial isolation and culture, can effectively identify pathogenic bacteria, solves the core pain points of traditional pathogen identification methods, can meet the needs of rapid diagnosis and precise medication for clinical infectious diseases, and can be widely used in the identification of bacterial pathogens in clinical medicine, public health and other fields.

[0094] Furthermore, the full-length Nanopore 16S sequencing data was analyzed using Emu software. The software analysis results output the relative abundance at the species / genus level, representing the proportion (a decimal of 0 and 1, convertible to a percentage) of each taxonomic unit (genus, species) in the sample relative abundance of all classifiable microorganisms. The relative abundance is an estimated proportion after iterative correction using a probabilistic model, specifically adapted to the full-length Nanopore 16S data of this invention, used to characterize the species composition structure of the sample's microbial community. As shown in Table 3, the method of this invention can characterize the bacterial species present in the sample and the relative abundance of each bacterial species.

[0095] discuss In addition to the method of this invention, there are many equivalent alternative technical solutions that can achieve the invention's objectives of rapid, culture-free, and accurate identification of clinical bacterial pathogens, shorten the detection cycle, guide precise drug use, and curb bacterial drug resistance. These alternatives can be made from three dimensions: target gene substitution, sequencing platform substitution, and detection technology route substitution. All of these can bypass the specific implementation methods limited by this invention and still achieve the same invention effect.

[0096] First, the target gene alternative. This invention uses the 16S rRNA gene as the core amplification and sequencing target. In addition, it can be replaced with bacterial conserved typing genes such as 23S rRNA and the 16S-23S rRNA interstitial region (ITS) as alternative amplification targets. These genes also possess bacterial species specificity and high conservation. Without the need for pure bacterial culture, pathogenic bacterial species can be identified directly from clinical samples through amplification and sequencing, followed by bioinformatics comparison and analysis. This also enables the detection of difficult-to-culture and unculturable bacteria, shortening the identification cycle and meeting the needs of rapid clinical pathogen screening, thus fully achieving the objectives of this invention.

[0097] Second, alternative sequencing and detection platforms. This invention uses 16S amplification-third-generation sequencing as its core technology route, which can be completely replaced by alternative sequencing platforms such as second-generation high-throughput sequencing (NGS); it can also be replaced by molecular detection technologies such as gene chip hybridization and targeted qPCR amplification panels. All of the above alternatives can directly detect nucleic acids in clinical blood, sputum, urine, and other samples without bacterial isolation and culture. This avoids the drawbacks of traditional culture methods, such as long cycles and missed or misdiagnosed cases, while enabling screening for unknown pathogens. It is suitable for rapid emergency testing and routine clinical screening scenarios, achieving the same diagnostic and public health application effects.

[0098] Third, an alternative overall technical approach. Metagenomic sequencing (mNGS) can be used as a complete alternative technical approach. It does not require targeted amplification of the 16S gene, but directly performs high-throughput sequencing and bioinformatics analysis on the nucleic acids of all microorganisms in clinical samples. It does not require bacterial culture and can simultaneously identify bacteria, rare fastidious bacteria, and unknown pathogens. It also has the advantages of high detection efficiency, wide identification range, and suitability for screening the causes of severe infections and sepsis. It can replace the technical approach of this invention to achieve the invention's objectives of rapid pathogen identification, guiding targeted drug use, reducing antibiotic abuse, and reducing mortality.

[0099] Based on the above alternatives, the technical points in the method of the present invention can be reasonably replaced, which is expected to yield other rapid, culture-free, and accurate identification methods for clinical bacterial pathogens, shortening the detection cycle, guiding precise drug use, and curbing bacterial drug resistance.

[0100] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A device for detecting bacterial infection in a sample, characterized in that, The device includes the following modules: (z1) Sample providing module, the sample providing module being configured to provide a sample to be tested; (z2) Amplification module, the amplification module being configured to amplify the sample to be tested using specific primer pairs, thereby obtaining amplification products; (z3) Sequencing module, the sequencing module being configured to: construct a sequencing library based on the amplification product and perform sequencing, thereby providing sequencing results; (z4) Result output module, the result output module being configured to: provide the bacterial infection status of the sample to be tested based on the sequencing results; The specific primer pair includes an upstream primer and a downstream primer, wherein the upstream primer contains the sequence shown in SEQ ID NO:1 and the downstream primer contains the sequence shown in SEQ ID NO:

2.

2. The apparatus as claimed in claim 1, characterized in that, The bacterial infection status of the sample includes: (i) Whether there is bacterial infection in the sample; (ii) The species of bacteria infecting the sample; (iii) The relative abundance ratio of each infecting bacterial species in the sample.

3. The apparatus as described in claim 1, characterized in that, The upstream primer is shown in SEQ ID NO:7, and the downstream primer is shown in SEQ ID NO:8; The sequencing in question refers to nanopore sequencing.

4. The apparatus as claimed in claim 1, characterized in that, The bacteria include: Mycobacterium tuberculosis, Streptococcus pneumoniae, Moraxella catarrhalis, Haemophilus influenzae, Legionella pneumophila, Klebsiella pneumoniae, Staphylococcus aureus, Group A beta-hemolytic streptococci, Pseudomonas aeruginosa, Acinetobacter baumannii, Escherichia coli, Neisseria meningitidis, Neisseria microflagellate, Corynebacterium stripe, or combinations thereof.

5. A reagent kit, characterized in that, The kit includes: (a) A primer pair comprising the sequence shown in SEQ ID NO:1 and the sequence shown in SEQ ID NO:2; (b) Amplification-essential reagents, wherein the amplification-essential reagents include: hot-start DNA polymerase, reverse transcriptase, dNTPs, magnesium ions, buffer, enzyme-free water, or combinations thereof.

6. The reagent kit as described in claim 5, characterized in that, The kit also contains: (c) Nanopore sequencing reagents, wherein the nanopore sequencing reagents include: RLB sample tag sequences, RAP adapters, magnetic beads, or combinations thereof.

7. Use of the kit according to claim 5 or 6, characterized in that, This is used to prepare a detection product for detecting bacterial infection in a sample and / or for broad-spectrum screening of samples with bacterial infection.

8. A method for detecting bacterial infection in a sample for non-diagnostic purposes, characterized in that, Includes the following steps: (S1) Provide the sample to be tested; (S2) The sample to be tested is amplified using a specific primer pair to obtain the amplification product; (S3) Construct a sequencing library based on the amplification primers and perform sequencing to provide sequencing results; (S4) Based on the sequencing results, provide the bacterial infection status of the sample to be tested; The specific primer pair includes an upstream primer and a downstream primer, wherein the upstream primer contains the sequence shown in SEQ ID NO:1 and the downstream primer contains the sequence shown in SEQ ID NO:

2.

9. The method as described in claim 8, characterized in that, The upstream primer is shown in SEQ ID NO:7, and the downstream primer is shown in SEQ ID NO:8; The sequencing in question refers to nanopore sequencing.

10. The method as described in claim 8, characterized in that, The samples to be tested include: food samples, environmental samples, or combinations thereof.

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