Bacterial priority pathogen detection primer group based on targeted sequencing, kit and application

Abstract

The invention discloses a bacterial priority pathogen detection primer group based on targeted sequencing, a kit and application, the primer group has the advantages of strong specificity, high sensitivity and the like through optimization design, can be combined with a multiple amplification system to simultaneously realize rapid and accurate detection of at most 14 bacterial priority pathogens, and effectively avoids false negative; the primer group can simultaneously amplify multiple pathogenic bacterium target genes in one reaction tube, and can efficiently detect clinical common pathogenic bacteria possibly existing in a sample in combination with a high-throughput sequencing technology, thereby providing a more accurate basis for diagnosis and treatment of respiratory tract infection.

Description

Technical Field

[0001] The present invention relates to the technical field of pathogen detection, and specifically to a primer set, a kit and an application for preferential pathogen detection of bacteria based on targeted sequencing. Background Art

[0002] Antimicrobial resistance (ABR) has been one of the biggest global public health and development threats. Since the World Health Organization (WHO) released its first list of priority pathogens for antibiotic-resistant bacteria in 2017, the threat of antimicrobial resistance has continued. In 2024, based on the 2017 list, WHO released the updated "Priority Pathogens List for Bacteria 2024", which contains 15 ABR pathogen families, classified into key, high, and medium priority categories for R&D and public health measures. In this update, WHO classified Gram-negative bacteria resistant to last-resort antibiotics, such as various pathogens in Acinetobacter baumannii and Enterobacteriaceae, and Mycobacterium tuberculosis resistant to rifampicin (RR), into the key priority category due to their ability to transfer resistance genes, the severity of the infections and diseases they cause, and the significant global burden they impose. Salmonella enterica (non-Typhi serovars), Salmonella enterica subsp. Typhi, and Shigella spp. were classified as high priority, reflecting their increasing resistance to existing treatments and the high infection burden associated with these pathogens. Other pathogens classified as high priority include antibiotic-resistant Pseudomonas aeruginosa and Staphylococcus aureus, as they pose a global threat, especially in the healthcare setting. The high priority category also covers pathogens that pose unique challenges to public health, such as Neisseria gonorrhoeae, in which multi-drug-resistant strains have emerged, limiting treatment options. Another pathogen of public health importance is antibiotic-resistant Enterococcus faecium, which is particularly important because it can spread resistance elements across the One Health spectrum. Streptococcus pyogenes group A, Streptococcus agalactiae group B, Streptococcus pneumoniae, and Haemophilus influenzae were included in the medium priority category, indicating the urgent need to address their impact on public health, especially on vulnerable populations in resource-limited settings. Timely identification of the pathogen type in patients with respiratory infections is crucial for further diagnosis and treatment work.Therefore, timely and accurate etiological diagnosis, early determination of the target pathogen, and targeted drug treatment are the keys to improving the prognosis of patients, reducing the case-fatality rate and the incidence of sequelae.

[0003] Traditional etiological detection methods, such as culture and immunological techniques, often take a long time and cannot cover all pathogens, resulting in many infections remaining undiagnosed. The metagenomics next-generation sequencing (mNGS) technology does not require the isolation and culture of pathogens and is not dependent on known nucleic acid sequences. It can detect various microorganisms without bias and with full coverage, and does not require specific amplification, greatly saving the detection time and improving the diagnostic efficiency. However, mNGS detection is a non-targeted detection, and the large amount of host nucleic acid in the sample may cause trace pathogen nucleic acid to be submerged under the host background, resulting in false negative results that cannot be detected.

[0004] Pathogen targeted sequencing (Targeted Next-Generation Sequencing, tNGS) can detect dozens to hundreds of known pathogenic microorganisms in the sample to be tested through the combination of ultra-multiplex PCR amplification and high-throughput sequencing. Compared with pathogen metagenomic sequencing (mNGS), tNGS has the advantages of a clear pathogen spectrum range and low sequencing cost.

[0005] Therefore, it is necessary to provide a primer set for the detection of priority bacterial pathogens based on targeted sequencing to improve the detection efficiency, specificity and sensitivity, and to achieve the simultaneous detection of multiple pathogens, providing a more accurate basis for the diagnosis and treatment of respiratory infections. Summary of the Invention

[0006] The present invention aims to develop a novel primer set and kit for targeted detection of pathogenic bacteria in respiratory infections. By optimizing primer design, the specificity, sensitivity and accuracy are improved to achieve rapid and accurate detection of multiple priority bacterial pathogens, providing a more accurate basis for the diagnosis and treatment of respiratory infections.

[0007] In view of this, the solution of the present invention is as follows:

[0008] The first aspect of the present invention is to provide a primer set for detecting bacterial priority pathogens based on targeted sequencing, which includes primer pairs with nucleotide sequences as shown in SEQ ID NO: 1-4, SEQ ID NO: 5-8, SEQ ID NO: 9-12, SEQ ID NO: 13-16, SEQ ID NO: 17-20, SEQ ID NO: 21-24, SEQ ID NO: 25-28, SEQ ID NO: 29-32, SEQ ID NO: 33-36, SEQ ID NO: 37-40, SEQ ID NO: 41-44, SEQ ID NO: 45-48, SEQ ID NO: 49-52 and SEQ ID NO: 53-56, respectively, for detecting Acinetobacter baumannii, Enterobacter, Salmonella typhi, Shigella, Enterococcus faecalis, Pseudomonas aeruginosa, non-typhoidal Salmonella, Neisseria gonorrhoeae, Staphylococcus aureus, Group A Streptococcus, Streptococcus pneumoniae, Haemophilus influenzae, Group B Streptococcus and Mycobacterium tuberculosis.

[0009] The second aspect of the present invention is to provide the application of the primer set described in the first aspect in the preparation of a kit for detecting bacterial priority pathogens.

[0010] The third aspect of the present invention is to provide a kit for detecting bacterial priority pathogens, which includes the primer set described in the first aspect and a sequencing adapter primer pair.

[0011] Furthermore, the nucleotide sequence of the sequencing adapter primer pair is as shown in SEQ ID NO: 57-58.

[0012] Furthermore, the kit also includes a PCR buffer and a DNA polymerase.

[0013] Preferably, the PCR buffer contains at least one of dNTPs, MgCl2, Tris-HCl, KCl and (NH4)2SO4.

[0014] Furthermore, the kit also includes a nucleic acid extraction reagent and / or a cDNA synthesis reagent. Preferably, the nucleic acid extraction reagent includes an enzymatic digestion reagent and / or a chemical lysis reagent.

[0015] The fourth aspect of the present invention is to provide a method for detecting bacterial priority pathogens for non-diagnostic purposes, using the primer set described in the first aspect, and the steps include:

[0016] S1. Obtaining the DNA and / or reverse transcription product cDNA of the sample to be tested;

[0017] S2. Using the DNA and / or cDNA as a template and performing PCR amplification with the primer set to obtain a product;

[0018] S3. Add the PCR amplification product to the sequencing adapter primer pair and perform PCR amplification again to obtain a sequencing library;

[0019] S4. Perform high-throughput sequencing on the sequencing library to obtain sequencing sequences;

[0020] S5. Compare the sequencing sequences with the pathogenic microorganism target sequence database, count the reads data of the targets, and calculate the normalized reads number of the pathogens.

[0021] Further, the sequencing adapter primer pair includes a forward universal primer and a reverse universal primer, which are respectively added to the 5' end of the forward primer and the 5' end of the reverse primer in the primer group.

[0022] Preferably, the nucleotide sequences of the forward universal primer and the reverse universal primer are shown in SEQ ID NO: 57 and 58 respectively, and are respectively connected to the 5' end of the odd-numbered sequences and the even-numbered sequences in the primer group described in SEQ ID NO: 1-56.

[0023] Further, the sample to be tested can be an environmental sample, such as water source, soil, air or daily necessities, etc., for detecting whether there are pathogenic microorganisms in the environment; the sample to be tested can also be a sample derived from animal secretions or an untraceable human-derived sample, including but not limited to samples such as bronchoalveolar lavage fluid, nasopharyngeal / oropharyngeal swabs, sputum, etc.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] The primer group for detecting priority pathogens of bacteria based on targeted sequencing provided by the present invention covers 14 common antibiotic-resistant bacteria, such as Acinetobacter baumannii, Escherichia coli, Salmonella typhi, Shigella spp., Enterococcus faecalis, Pseudomonas aeruginosa, non-typhoidal Salmonella, Neisseria gonorrhoeae, Staphylococcus aureus, Group A Streptococcus, Streptococcus pneumoniae, Haemophilus influenzae, Group B Streptococcus and Mycobacterium tuberculosis. Through optimized primer design, the primer group has the advantages of strong specificity and high sensitivity, and can simultaneously achieve rapid and accurate detection of up to 14 bacterial priority pathogens in combination with a multiplex amplification system, effectively avoiding false negatives.

[0026] The kit provided by the present invention contains a multiplex primer system, which can simultaneously amplify multiple pathogenic bacterial target genes in one reaction tube. Combined with high-throughput sequencing technology, it can efficiently detect clinically common pathogenic bacteria that may exist in the sample, providing a more accurate basis for the diagnosis and treatment of respiratory infections. Description of the Drawings

[0027] Figure 1 This is the comparison result of the amplification effect before primer optimization in Example 2 of the present invention.

[0028] Figure 2 This is the comparison result of the amplification effect after the primer optimization in Example 2 of the present invention.

[0029] Figure 3 This is the amplification effect performance of Acinetobacter baumannii before the single-system primer optimization in Example 2 of the present invention.

[0030] Figure 4 This is the amplification effect performance of Acinetobacter baumannii after the single-system primer optimization in Example 2 of the present invention.

[0031] Figure 5 This is the electrophoresis detection result of the multiplex PCR amplification product in Example 2 of the present invention. Detailed implementation manners

[0032] Next, the technical solutions of the present invention will be clearly and completely described in conjunction with the preferred embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0033] Example 1 Selection and determination of pathogenic bacteria

[0034] According to the updated catalog of pathogenic bacteria of the World Health Organization, the present application has determined the targeted amplification for 14 drug-resistant bacteria in the catalog.

[0035] The 14 bacterial pathogens are shown in Table 1:

[0036]

[0037]

[0038] Example 2 Target region screening and primer design

[0039] 1) Screening of target regions

[0040] After determining the types of pathogenic microorganisms, to achieve specificity and sensitivity in the multiplex detection process, when designing the targeted primers, the logical consideration for the target regions is that the specificity can reach more than 94% and the coverage can reach more than 94%.

[0041] 2) Primer design and screening

[0042] In view of the fact that the present application is a multiplex primer system, the primer design requirements of the present application are strict, and many factors need to be considered. Generally, the following steps are experienced:

[0043] a) Preliminary design of multiplex PCR primers using primer design software or primer design websites; Requirements: The length of the primer amplicon is 100 - 500 bp, the GC content of the sequence is 45% - 65%, the primer length is 17 - 25 bp, there is no base complementarity between the two primers, and the primer does not have a poly sequence with more than 5 consecutive bases.

[0044] b) Re-screen the primers through bioinformatics analysis to select primers with high specificity and low dimer formation;

[0045] c) Conduct wet experiments to screen out multiplex PCR primers with high amplification efficiency and adjust the specific primer sequences accordingly.

[0046] When the primers of this application are used, the 5' end of each forward amplification primer is connected to the forward universal primer; the 5' end of each reverse amplification primer is connected to the reverse universal primer. Among them, the forward amplification primer is the sequence that recognizes the pathogen target and amplifies, and the sequences of the forward universal primer and the reverse universal primer are the adapter sequences of the Illumina sequencing platform. The sequences of the forward universal primer and the reverse universal primer are as follows:

[0047] F: 5'-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-3' (SEQ ID NO: 57)

[0048] R: 5'-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-3' (SEQ ID NO: 58)

[0049] The optimization process of the primer sequence includes target region optimization and single amplification optimization (optimizing primer Tm value and GC content), and the target region optimization is shown in Table 1.

[0050] Table 1:

[0051]

[0052] Due to the large number of primers, taking Acinetobacter baumannii (A.baumannii) as an example, the primers in the initial design and after optimization are shown in Table 2.

[0053] Table 2:

[0054]

[0055] Through the detection of the amplification efficiency of single primers, it is found that some primers before optimization have low amplification efficiency or low primer specificity, as Figure 1 shown, and they cannot meet the mixing requirements of multiplex PCR. After re-designing and optimizing the sequences through bioinformatics analysis, the amplification efficiency and specificity of the primers are significantly improved, as Figure 2As shown, the adjusted primers have a significant advantage in amplification efficiency. Figure 1-2 The corresponding relationships of the electrophoresis bands in Figure 1-2 are as follows: Bands numbered 1-2 correspond to Acinetobacter baumannii, 3-4 to Enterobacteriaceae, 5-6 to Salmonella enterica subsp. Typhi, 7-8 to Shigella spp., 9-10 to Enterococcus faecium, 11-12 to Pseudomonas aeruginosa, 13-14 to Salmonella enterica (non-Typhi serovars), 15-16 to Neisseria gonorrhoeae, 17-18 to Staphylococcus aureus, 19-20 to Streptococcus pyogenes group A, 21-22 to Streptococcus pneumoniae, 23-24 to Haemophilus influenzae, 25-26 to Streptococcus agalactiae group B, and 27-28 to Mycobacterium tuberculosis electrophoresis bands. The two bands in each group respectively correspond to the amplification results of different primer pairs.

[0056] As Figure 3 shown, taking Acinetobacter baumannii as an example, the amplification effect before the optimization of the single-system primers was poor, and the amplification effect after optimization is as Figure 4 shown. The adjusted primers have a significant advantage in amplification efficiency.

[0057] c. Optimization of the multiplex system

[0058] After individual screening, the mixed primer sets are subjected to multiplex amplification. For Streptococcus pneumoniae, in the initial design, it could not be amplified in the multiplex system; after the optimization of the primer sets, it could be effectively amplified in the multiplex system, as Figure 5 shown, where: Figure A is the analysis result of the amplification products of two pairs of primers before primer optimization; Figure B is the analysis result of the amplification products of two pairs of primers after primer optimization. The sequencing results show that Streptococcus pneumoniae was effectively amplified after optimization and adjustment, as shown in Table 3 below.

[0059] Table 3:

[0060]

[0061] Based on the above optimization experiments, the finally determined primer system is shown in Table 4. Through the optimization of the primers, each pair of primers can be effectively amplified in the multiplex system.

[0062] Table 4:

[0063]

[0064]

[0065]

[0066] Example 3 Preparation of Kit and Establishment of Detection System

[0067] Based on the above primer amplification system, a kit of the present application is established. The kit includes the above primer system and also includes a PCR buffer and a DNA polymerase. Exemplarily, the PCR buffer can be any buffer that can complete PCR amplification, including but not limited to dNTPs, MgCl2, Tris-HCl, KCl, and (NH4)2SO4, etc. The kit can also include a sample DNA / RNA co-extraction reagent and a reverse transcription reagent; Exemplarily, the sample DNA / RNA extraction reagent can be any reagent, combination, or kit that can extract nucleic acids, such as enzymatic and chemical reagent lysis methods, by digesting proteins and lysing cells with enzymes (such as proteinase K) and chemical reagents (such as guanidine isothiocyanate). Among them, the enzyme includes but is not limited to proteinase K, and the chemical reagent includes but is not limited to guanidine isothiocyanate.

[0068] Establish a pathogen detection method based on targeted sequencing, which specifically includes the following steps:

[0069] 1. Obtain the nucleic acid of the sample to be tested;

[0070] This process uses the ZYMO Quick-DNA / RNA TM Viral Kit kit (D7020) to extract the nucleic acid (DNA and RNA) of the sample to be tested. The operation steps are as follows:

[0071] (1) Prepare 0.5% (V / V) β-mercaptoethanol solution and 80% absolute ethanol for later use;

[0072] (2) At room temperature (20 - 30 °C), add the DNA / RNA Shield protective solution to the sample in a 1:1 ratio;

[0073] (3) Add 400 μL of Viral DNA / RNA Buffer to 400 μL of the sample and mix well;

[0074] (4) Transfer the mixture to the adsorption column Zymo-Spin TM IIC-XLR Column, place it in the collection tube, centrifuge at 14000g for 2 min, and transfer the adsorption column to a new collection tube;

[0075] (5) Add 500 μL of Viral Wash Buffer to the adsorption column, centrifuge at 12,000 g for 30 sec, discard the waste liquid, and repeat this step;

[0076] (6) Add 500 μL of 80% absolute ethanol to the adsorption column, centrifuge at 12,000 g for 1 min to ensure complete removal of the eluate. Replace the collection tube with a new one, centrifuge at 12,000 g for 1 min with empty spin, and carefully transfer the column to a new 1.5 mL nuclease-free centrifuge tube;

[0077] (7) Suspend and add 35 μL of Nuclease-Free Water (Ambion TM , Invitrogen) to the middle position of the adsorption membrane. After standing at room temperature for 1 min, centrifuge at 14,000 g for 1 min. Retain the eluate in the 1.5 mL centrifuge tube and discard the adsorption column;

[0078] (8) Take 1 μL of the elution product for Qubit quantification (Thermo Fisher) to determine the concentration of the extracted nucleic acid; if the extraction concentration is 0, resample and extract.

[0079] 2. Using DNA as a template, perform multiplex PCR amplification respectively with the primer sets provided in Table 4, and prepare a product mixture from the DNA amplification product and the cDNA amplification product in a certain proportion;

[0080] (1) Add each component to an eight-strip tube according to the system described in Table 5 for PCR amplification;

[0081] Table 5:

[0082] Component Volume (μL) Enzyme Premix (Tiangen) 12.5 Primer Set F 1.0 Primer Set R 1.0 Nucleic Acid Template 10.0 Nuclease-Free Water 0.5 Total 25.0

[0083] (2) Set the program described in Table 6 on the PCR instrument for PCR amplification;

[0084] Table 6:

[0085]

[0086] 3. Using the product mixture obtained in step 2 as a template, perform adapter-added PCR amplification with the adapter sequence primer pairs described in SEQ ID NO: 57 - 58 to add sequencing adapter sequences to the multiplex PCR amplification products and obtain a sequencing library;

[0087] (1) Add each component to an eight-strip tube according to the system described in Table 7 for PCR amplification;

[0088] Table 7:

[0089] Component Volume (μL) Enzyme Premix (Tiangen) 12.5 Adapter Universal Primer Index 2.0 Product of Step 2 10.0 Nuclease-Free Water 0.5 Total 25.0

[0090] (2) Set the program as described in Table 8 on a PCR instrument for PCR amplification;

[0091] Table 8:

[0092]

[0093] (3) Purify the above PCR products with magnetic beads (Agencout AMPure XP, Beckman), and the final elution volume is 30 μL.

[0094] 4. Perform high-throughput sequencing on the sequencing library obtained in Step 3 to obtain sequencing sequences;

[0095] The sequencing system used in this step is the NEXTSeq 1000 / 2000 sequencer. Just perform the on-machine sequencing according to the corresponding operation steps. After about 8 h, the sequencing sequence results are obtained.

[0096] 5. Compare the sequencing sequences with the pathogen microorganism target sequence database, and count the number of reads that accurately align to the specific pathogenic bacteria target;

[0097] 6. Calculate the normalized reads number of pathogen microorganisms using the following formula:

[0098]

[0099] Among them, RPM represents the number of pathogen microorganisms detected per million reads; Pathogen reads number refers to the number of reads with pathogen detection; Clean reads number represents the number of effective reads obtained after sample pretreatment.

[0100] Example 4 Detection Limit Performance Evaluation

[0101] The final results of the performance evaluation of 14 bacterial pathogens are shown in Table 9. The minimum detection limit of all species can reach 10 CFU / mL.

[0102] Table 9:

[0103]

[0104] Example 5 Clinical Sample Detection Verification

[0105] Collect 19 clinical samples with known pathogen conditions. Detect them by the method of this application and compare with the known standards. The results are shown in Table 10, and the predicted accuracy rate is 100%.

[0106] Table 10:

[0107]

[0108] Although embodiments of the present invention have been shown and described, it will be understood by those of ordinary skill in the art that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of the present invention, and the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A primer set for detecting bacterial priority pathogens based on targeted sequencing, characterized in that, Primer pairs containing nucleotide sequences as shown in SEQ ID NO: 1-4, SEQ ID NO: 5-8, SEQ ID NO: 9-12, SEQ ID NO: 13-16, SEQ ID NO: 17-20, SEQ ID NO: 21-24, SEQ ID NO: 25-28, SEQ ID NO: 29-32, SEQ ID NO: 33-36, SEQ ID NO: 37-40, SEQ ID NO: 41-44, SEQ ID NO: 45-48, SEQ ID NO: 49-52, and SEQ ID NO: 53-56 are respectively used for the detection of Acinetobacter baumannii, Enterobacter, Salmonella typhi, Shigella, Enterococcus faecalis, Pseudomonas aeruginosa, non-typhoidal Salmonella, Neisseria gonorrhoeae, Staphylococcus aureus, Group A Streptococcus, Streptococcus pneumoniae, Haemophilus influenzae, Group B Streptococcus, and Mycobacterium tuberculosis.

2. Use of the primer set according to claim 1 in the preparation of a kit for detecting bacterial priority pathogens.

3. Bacterial Priority Pathogen Detection Kit, characterized in that, It includes the primer set according to claim 1 and a sequencing adapter primer pair.

4. The kit according to claim 3, characterized in that, The nucleotide sequence of the sequencing adapter primer pair is as shown in SEQ ID NO: 57-58.

5. The kit according to claim 3, wherein The kit further includes a PCR buffer and a DNA polymerase.

6. The kit according to claim 5, wherein The PCR buffer contains at least one of dNTPs, MgCl2, Tris-HCl, KCl, and (NH4)2SO4.

7. The kit according to claim 3, characterized in that, It also includes a nucleic acid extraction reagent and / or a cDNA synthesis reagent.

8. The kit according to claim 7, characterized in that, The nucleic acid extraction reagent includes an enzymatic digestion reagent and / or a chemical lysis reagent.

9. A method for detecting bacterial priority pathogens for non-diagnostic purposes, using the primer set according to claim 1, characterized in that the steps It includes: S1. Obtain the DNA of the sample to be tested and / or the reverse transcription product cDNA; S2. Using the DNA and / or cDNA as a template, perform PCR amplification with the primer set to obtain a product; S3. Add the sequencing adapter primer pair to the PCR amplification product and perform PCR amplification again to obtain a sequencing library; S4. Perform high-throughput sequencing on the sequencing library to obtain sequencing sequences; S5. Compare the sequencing sequences with the pathogen target sequence database, count the reads data of the targets, and calculate the normalized reads number of the pathogens.

10. The application according to claim 9, characterized in that, The sequencing adapter primer pair includes a forward universal primer and a reverse universal primer, which are respectively added to the 5' end of the forward primer and the 5' end of the reverse primer in the primer set.

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