Primer group and method for escherichia coli MLST typing based on hospital localized microorganism mNGS platform

Abstract

The invention belongs to the technical field of microbiological detection, and particularly relates to an escherichia coli MLST typing primer group and method based on a hospital localized microbiological mNGS platform. The primer group comprises a primer pair for amplifying a dinB gene, an icdA gene, a pabB gene, a polB gene, a putP gene, a trpB gene, a trpA gene and an uriA gene, and corresponding nucleotide sequences are as shown in SEQ ID NO: 1-16. The primer group comprises a primer pair for amplifying the dinB gene, the icdA gene, the pabB gene, the polB gene, the putP gene, the trpB gene, the trpA gene and the uriA gene. According to the technical characteristics of short sequencing read length (50 bp) and single-ended sequencing of a next-generation sequencing platform in a hospital, an amplification primer group for the escherichia coli MLST housekeeping gene is redesigned, and amplicons are adaptive to the sequencing working principle of the existing next-generation sequencing platform in the hospital by optimizing the lengths and positions of the amplicons. By utilizing the primer group, the kit or the method, the genetic relationship between hospital infection pathogenic bacteria can be quickly and accurately judged (within 24 hours), the timeliness and scientificity of infection prevention and control are remarkably improved, and the primer group, the kit or the method have a wide clinical application prospect.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbial detection, and in particular, relates to a primer set and method for MLST typing of Escherichia coli based on a hospital-based microbial mNGS platform. Background Art

[0002] Nosocomial infections, also known as hospital-acquired infections, refer to illnesses that develop while patients are receiving treatment or care in a hospital. Hospitals should proactively implement measures to prevent and control nosocomial infections to safeguard patient health and safety. Early detection and treatment are crucial. Only by promptly identifying signs of infection and implementing effective measures can the impact of infection on patients be minimized and its further spread prevented. To determine whether a nosocomial infection has occurred, bacteria must be "typed." This is determined by whether the bacteria are of the same type, providing a basis for subsequent isolation, tracing, and other measures.

[0003] Currently, there are multiple methods used for microbial typing, including pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), and whole genome sequencing (WGS). MLST, with its advantages of high accuracy and uniform standards, is currently the most commonly used method. MLST is a typing technique based on nucleic acid sequences at multiple conserved gene loci, which are internationally recognized and known. MLST relies on differences in conserved bacterial gene sequences for typing, and therefore consists of a two-step process: sequencing and database comparison. The type corresponding to each gene is mapped to an overall "ST" type.

[0004] In practice, sequence analysis of multiple conserved gene loci reveals that if multiple patients in the same ward share the same ST-type of bacteria, this suggests the possibility of nosocomial infection or explosive spread. Conversely, if the ST-types of bacteria infecting different patients vary significantly, this can rule out the possibility of close transmission between bacteria and, consequently, nosocomial infection. Therefore, bacterial typing is an important basis for determining nosocomial transmission or overall epidemic trends.

[0005] The existing MLST typing relies on first-generation sequencing (Sanger sequencing) technology. After the bacteria are isolated, the hospital staff first needs to perform PCR amplification based on the gene combination of each bacterial housekeeper, and then send the amplified product to the sequencing company for testing. After obtaining the data, it is uploaded to an overseas website for query and finally the ST type is determined. This process has the following disadvantages: (1) Equipment and technology limitations: Hospitals and CDCs generally do not have relevant equipment and technology, so they can only be sent to third-party institutions for testing. The delivery process consumes logistics time, which seriously affects timeliness. (2) Result delivery and manual operation: After sequencing is completed, the test results are delivered via the website or email, but the final splicing and comparison work still requires manual operation, which is time-consuming and labor-intensive and prone to errors. (3) Timeliness: The entire process of amplification, first-generation sequencing, determination of bacterial ST typing, and data collation needs to be at least solid and reliable, but the second-generation sequencing used for scientific research also has the problem of sending samples for sequencing, and the sequencing cycle is as long as ~3 days, which does not meet the actual needs of clinical infection prevention and control. (4) Data security: The judgment and query of ST type require uploading data to an overseas website, which poses a risk of data leakage. On another level, from a scientific research perspective, there is also a method for cgMLST analysis using whole-genome sequencing. However, this method produces a large amount of data, with results taking 7 to 15 days to arrive. It is only suitable for retrospective studies and cannot provide timely guidance for clinical hospital infection control.

[0006] Therefore, when conducting bacterial MLST typing required for hospital infection prevention and control, although the first-generation sequencing technology has poor timeliness, complex operation, and information leakage, it is still the main method currently used.

[0007] To diagnose patients with difficult infectious diseases within 24 hours, some hospitals are actively developing localized next-generation sequencing platforms, enabling them to perform pathogen sequencing services within the hospital (rather than outsourcing to sequencing companies). While the purpose of building local sequencing platforms is to detect pathogens, they also provide the material foundation and technical support for leveraging these platforms to conduct homology analysis of nosocomial infections through technological innovation. Summary of the Invention

[0008] The purpose of the present invention is to provide a primer set and method for MLST typing of Escherichia coli based on a hospital-based microbial mNGS platform, so as to achieve rapid and accurate typing detection of Escherichia coli.

[0009] To this end, the present invention provides the following technical solutions.

[0010] The first aspect of the present invention provides a primer set for MLST typing of Escherichia coli based on a hospital-based microbial mNGS platform, comprising amplification dinB Primer pairs for gene amplification icdAPrimer pairs for gene amplification pabB Primer pairs for gene amplification polB Primer pairs for gene amplification putP Primer pairs for gene amplification trpB Primer pairs for gene amplification trpA Primer pairs and amplification of genes uidA primers for genes; The amplification dinB The primer pair for the gene includes a forward primer Ecoli1_dinB-F having a nucleotide sequence as shown in SEQ ID NO: 1, and a reverse primer Ecoli1_dinB-R having a nucleotide sequence as shown in SEQ ID NO: 2; The amplification icdA The primer pair for the gene includes a forward primer Ecoli2_icdA-F having a nucleotide sequence as shown in SEQ ID NO: 3, and a reverse primer Ecoli2_icdA-R having a nucleotide sequence as shown in SEQ ID NO: 4; The amplification pabB The primer pair for the gene includes a forward primer Ecoli3_pabB-F having a nucleotide sequence as shown in SEQ ID NO: 5, and a reverse primer Ecoli3_pabB-R having a nucleotide sequence as shown in SEQ ID NO: 6; The amplification polB The primer pair for the gene includes a forward primer Ecoli4_polB-F having a nucleotide sequence as shown in SEQ ID NO: 7, and a reverse primer Ecoli4_polB-R having a nucleotide sequence as shown in SEQ ID NO: 8; The amplification putP The primer pair for the gene includes a forward primer Ecoli5_putP-F having a nucleotide sequence as shown in SEQ ID NO: 9, and a reverse primer Ecoli5_putP-R having a nucleotide sequence as shown in SEQ ID NO: 10; The amplification trpB The primer pair for the gene includes a forward primer Ecoli6_trpB-F having a nucleotide sequence as shown in SEQ ID NO: 11, and a reverse primer Ecoli6_trpB-R having a nucleotide sequence as shown in SEQ ID NO: 12; The amplification trpA The primer pair for the gene includes a forward primer Ecoli7_trpA-F having a nucleotide sequence as shown in SEQ ID NO: 13, and a reverse primer Ecoli7_trpA-R having a nucleotide sequence as shown in SEQ ID NO: 14; and The amplification uidAThe primer pair for the gene includes a forward primer Ecoli8_uidA-F whose nucleotide sequence is shown in SEQ ID NO: 15, and a reverse primer Ecoli8_uidA-R whose nucleotide sequence is shown in SEQ ID NO: 16.

[0011] A second aspect of the present invention provides a kit for MLST typing of Escherichia coli based on a hospital-localized microbial mNGS platform, the kit comprising the primer set as described above.

[0012] In a preferred embodiment of the present invention, the kit further comprises 2X TaqMan Fast qPCR MasterMix.

[0013] In a preferred embodiment of the present invention, the concentration of each primer in the kit is independently 10 μM.

[0014] The third aspect of the present invention provides a use of the primer set or kit as described above in constructing an E. coli mNGS sequencing library for MLST typing and tracing of E. coli.

[0015] A fourth aspect of the present invention provides an mNGS sequencing library construction method for MLST typing and tracing of Escherichia coli, comprising the following steps: 1) Extract DNA from the sample to be tested; 2) performing qPCR amplification on the DNA of the sample extracted in step 1) using the kit described above; 3) Add mNGS sequencing adapters to both ends of the amplification reaction product of step 2) to obtain an mNGS sequencing library.

[0016] In a preferred embodiment of the present invention, in step 1), a bacterial genomic DNA extraction kit is used to extract DNA from the sample to be tested.

[0017] In a preferred embodiment of the present invention, in step 2), the reaction conditions for the qPCR amplification reaction of the test sample DNA are: 94°C for 2 min, cycle number 1; 94°C for 30 s, cycle number 35; 50°C for 1 min; 72°C for 30 s; 72°C for 5 min, cycle number 1; 4°C forever.

[0018] In a preferred embodiment of the present invention, in step 2), the reaction system for qPCR amplification of the sample DNA to be tested comprises: In a preferred embodiment of the present invention, the instrument used for mNGS sequencing is PanGenomics 2000, and the detection server used is CentOS Linux release 7.9.2009 (Core).

[0019] A fifth aspect of the present invention provides a use of the primer set, kit, or method as described above in preparing a reagent for detecting MLST typing and tracing of Escherichia coli.

[0020] By means of the above technical solution, the present invention has at least the following advantages: Based on the technical characteristics of short sequencing read length and single-end sequencing of the second-generation sequencing platform in the hospital, the present invention redesigns the primers for amplifying the Escherichia coli MLST housekeeping gene. By extending the amplicon length, it is intended to improve the multi-locus sequence analysis (MLST) technology, making it suitable for the hospital's existing second-generation sequencing platform. Using the products amplified by the primer set of the present invention to build a library and sequence them using a specific second-generation sequencing platform, rapid and accurate homology analysis of Escherichia coli can be achieved, assisting in the prevention and control of nosocomial infections. Utilizing the primer set, kit or method of the present invention, it is possible to quickly (within 24 hours) and accurately determine the kinship between pathogens of nosocomial infections, significantly improving the timeliness and scientific nature of infection prevention and control, reducing the incidence of nosocomial infections, reducing patient hospitalization time and medical expenses, and improving patient safety and hospital reputation in the long term, and has broad clinical application prospects.

[0021] By optimizing primer design and sequencing workflows, this invention enables the use of remaining chip positions for bacterial MLST typing in parallel with routine mNGS testing in daily clinical practice. This not only improves testing efficiency but also reduces costs while maintaining high accuracy and timeliness. This provides a more efficient and cost-effective bacterial typing solution for hospitals and CDCs.

[0022] The primer set, kit and corresponding sequencing method of the present invention can also promote inter-regional cooperation in hospital infection prevention and control, improve regional public health levels, and lay the foundation for subsequent technology upgrades and wider clinical applications. They have important academic value and social significance for improving medical quality and promoting public health development.

[0023] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention and implement it according to the contents of the specification, the following is a detailed description of the preferred embodiments of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1The colony morphology of two Escherichia coli strains, NJGLYY_4758 and NJGLYY_2254, is shown; the upper figure shows the colony morphology of the strain cultured on blood agar at 35°C in 5% CO2 for 24 hours; the lower figure shows the colony morphology of the strain cultured on MH agar at 35°C for 24 hours; Figure 2 The figure shows the typing result of strain NJGLYY4758 in Example 2; Figure 3 The figure shows the typing result of strain NJGLYY2254 in Example 2; Figure 4 The figure shows the quality analysis of the whole genome sequencing of strain NJGLYY4758 in Example 3; Figure 5 The quality analysis graph of the whole genome sequencing of strain NJGLYY2254 in Example 3 is shown; Figure 6 The figure shows the results of whole-genome sequencing analysis of strain NJGLYY4758 in Example 3; Figure 7 The figure shows the results of whole-genome sequencing analysis of strain NJGLYY2254 in Example 3. DETAILED DESCRIPTION

[0025] In order to make the technical means, creative features, objectives and effects achieved by the present invention easier to understand, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts shall fall within the scope of protection of the present invention.

[0026] Traditional MLST technology is based on first-generation sequencing, and its primer design positions and amplicon lengths fully take into account the technical characteristics of first-generation sequencing. However, the present invention redesigns primers and adjusts amplicon length based on the characteristics of the second-generation sequencing platform.

[0027] Therefore, in one embodiment, the present invention provides a primer set for MLST typing of Escherichia coli based on a hospital-based microbial mNGS platform, comprising amplification dinB Primer pairs for gene amplification icdA Primer pairs for gene amplification pabB Primer pairs for gene amplification polB Primer pairs for amplifying putP genes, primer pairs for amplifying trpB Primer pairs for gene amplification trpA Primer pairs and amplification of genes uidA primer pairs for genes; The amplification dinB The primer pair for the gene includes a forward primer Ecoli1_dinB-F having a nucleotide sequence as shown in SEQ ID NO: 1, and a reverse primer Ecoli1_dinB-R having a nucleotide sequence as shown in SEQ ID NO: 2; The amplification i The primer pair for the cdA gene includes a forward primer Ecoli2_icdA-F having a nucleotide sequence as shown in SEQ ID NO: 3, and a reverse primer Ecoli2_icdA-R having a nucleotide sequence as shown in SEQ ID NO: 4; The amplification pabB The primer pair for the gene includes a forward primer Ecoli3_pabB-F having a nucleotide sequence as shown in SEQ ID NO: 5, and a reverse primer Ecoli3_pabB-R having a nucleotide sequence as shown in SEQ ID NO: 6; The amplification polB The primer pair for the gene includes a forward primer Ecoli4_polB-F having a nucleotide sequence as shown in SEQ ID NO: 7, and a reverse primer Ecoli4_polB-R having a nucleotide sequence as shown in SEQ ID NO: 8; The amplification putP The primer pair for the gene includes a forward primer Ecoli5_putP-F having a nucleotide sequence as shown in SEQ ID NO: 9, and a reverse primer Ecoli5_putP-R having a nucleotide sequence as shown in SEQ ID NO: 10; The amplification trpB The primer pair for the gene includes a forward primer Ecoli6_trpB-F having a nucleotide sequence as shown in SEQ ID NO: 11, and a reverse primer Ecoli6_trpB-R having a nucleotide sequence as shown in SEQ ID NO: 12; The amplification trpA The primer pair for the gene includes a forward primer Ecoli7_trpA-F having a nucleotide sequence as shown in SEQ ID NO: 13, and a reverse primer Ecoli7_trpA-R having a nucleotide sequence as shown in SEQ ID NO: 14; and The amplification uidA The primer pair for the gene includes a forward primer Ecoli8_uidA-F whose nucleotide sequence is shown in SEQ ID NO: 15, and a reverse primer Ecoli8_uidA-R whose nucleotide sequence is shown in SEQ ID NO: 16.

[0028] For example, the specific sequence information of the above primer set is shown in the following table: The workload can be greatly reduced by constructing multiple amplification primers. When designing multiple amplification primers, cross-reactions or nonspecific amplifications between primers should be avoided to the greatest extent possible. In addition, amplification conditions, such as primer concentration, annealing temperature, etc., need to be optimized to ensure the consistency of target gene amplification efficiency. Overall, through bioinformatics prediction and experimental optimization, a set of efficient and stable multiple amplification primer combinations is constructed to provide a qualified DNA library for sequencing, which is another key technological innovation of this application.

[0029] In another embodiment, a kit for MLST typing and tracing of Escherichia coli is provided, wherein the kit comprises the above-mentioned primer set.

[0030] Preferably, the kit further comprises 2X TaqMan Fast qPCR Master Mix.

[0031] In a further optimized example, the concentration of each primer in the kit was independently 10 µM.

[0032] In another embodiment, there is provided a use of the primer set or kit as described above in constructing an mNGS sequencing library for E. coli MLST typing and tracing.

[0033] By applying the primer set and kit to construct mNGS sequencing libraries for E. coli MLST typing and tracing, the system is fully compatible with existing next-generation sequencing platforms, eliminating the need for additional equipment and ensuring efficient operation under existing conditions. Bacterial homology analysis can be completed within 24 hours, meeting the timeliness requirements of hospital-acquired infection prevention and control. Its accuracy is comparable to that of traditional MLST or WGS methods.

[0034] In another embodiment, a method for constructing an mNGS sequencing library for MLST typing and tracing of Escherichia coli is provided, comprising the following steps: 1) Extract DNA from the sample to be tested; 2) performing qPCR amplification on the DNA of the sample extracted in step 1) using the kit described above; 3) Add mNGS sequencing adapters to both ends of the amplification reaction product of step 2) to obtain an mNGS sequencing library.

[0035] In step 1), DNA is extracted from the sample to be tested using a bacterial genomic DNA extraction kit.

[0036] In step 2), the reaction conditions for qPCR amplification of the test sample DNA are as follows: 94°C for 2 min, cycle number 1; 94°C for 30 s, cycle number 35; 50°C for 1 min; 72°C for 30 s; 72°C for 5 min, cycle number 1; 4°C forever.

[0037] In step 2), the reaction system for qPCR amplification of the sample DNA to be tested is composed of: The primer sets, kits, and methods used in this invention are particularly suitable for mNGS sequencing using the PanGenomics 2000 instrument and the CentOS Linux release 7.9.2009 (Core) detection server. Combining these methods can shorten bacterial typing time and increase accuracy.

[0038] The following embodiments relate to and refer to: 1. mNGS detection platform for E. coli MLST typing and traceability: The instrument used is PanGenomics 2000, and the configured server is shown in Table 1.

[0039] Table 1 Instrument Model Example 1: Design of primer sets The present invention is based on the traditional MLST system, and the housekeeping genes of Escherichia coli are: putP、icdA、trpA、pabB、 polB, dinB, trpB, uidA (PubMLST: https: / / pubmlst.org / data). Therefore, based on the technical characteristics of the in-house next-generation sequencing platform (short sequencing read length, single-end sequencing), MLST housekeeping gene amplification primers were redesigned to extend the amplicon length. During the primer design process, attention was paid to optimizing parameters such as primer length, GC content, and Tm value to ensure amplification efficiency and sequencing quality.

[0040] For amplification specificity, first use the Primer-BLAST website and select the "nr" database (the standard non-redundant database) to comprehensively check whether there is non-specific amplification between species; after confirming the species specificity, further use the Primer-BLAST website and select " Escherichia coli "As the target species, check the non-specific amplification in the species. If there is no non-specific amplification, it is the appropriate primer. Based on the above principles and optimized screening, the amplification was finally obtained. dinBPrimer pairs for gene amplification icdA Primer pairs for gene amplification pabB Primer pairs for gene amplification polB Primer pairs for gene amplification putP Primer pairs for gene amplification trpA Primer pairs for gene amplification trpB Primer pairs and amplification of genes uidA The primer pairs for the genes, the sequences of the primer sets are shown in SEQ ID NOs: 1 to 16, and the specific nucleotide sequences and amplified fragment information are shown in Table 2: Table 2 PCR amplification primer sets Example 2: MLST typing detection of Escherichia coli In this example, two strains of Escherichia coli isolated from the blood of a subject were used as test samples, and the primer set determined in Example 1 was used to perform MLST typing on the test samples, as follows: 1.1 Strain source and information The strains used in this example were isolated from the blood of patients with cholangitis and systemic lupus erythematosus who visited Nanjing Drum Tower Hospital. Their specific information is shown in Table 3: Table 3 Escherichia coli strain information Figure 1 The culture morphology of two strains, NJGLYY_4758 and NJGLYY_2254, is shown; the upper figure is a colony morphology image of the culture on blood agar medium at 35°C and 5% CO2 for 24 hours; the lower figure is a colony morphology image of the culture on MH agar medium at 35°C for 24 hours.

[0041] 1.2 Extraction of genomic DNA Bacterial genomic DNA was extracted using a bacterial genomic DNA extraction kit (DP302, purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.) according to the operating instructions to obtain a DNA solution of the corresponding strain.

[0042] 1.3 PCR amplification Subsequently, the primer set shown in Table 2 of Example 1 was used to perform PCR amplification reaction on the DNA of the strain to be tested, wherein the composition of the 20 μL reaction system is shown in Table 4: Table 4 PCR amplification reaction system Note: 2X TaqMan Fast qPCR Master Mix in the table, catalog number: B639274, was purchased from Sangon Biotech (Shanghai) Co., Ltd.

[0043] The reaction program was: 94°C for 2 min, cycle 1; 94°C for 30 s, cycle 35; 50°C for 1 min; 72°C for 30 s; 72°C for 5 min, cycle 1; 4°C forever.

[0044] The PCR amplification reaction was performed according to the above reaction system and procedure to obtain the PCR reaction product.

[0045] 1.4 Product purification The obtained PCR reaction product is purified, specifically comprising the following steps: Pipette 45 μL of DNA purification magnetic beads into 50 μL of PCR product. Vortex or pipette 10 times to mix thoroughly. Incubate at room temperature for 5 minutes. Briefly centrifuge to collect the mixture at the bottom of the tube. Place the PCR tube on a magnetic rack and let it sit for approximately 3-5 minutes. Once the liquid in the tube is clear, carefully aspirate and discard. Add 200 μL of 80% ethanol (freshly prepared immediately before use), let it sit for 30 seconds, and carefully aspirate and discard the liquid. It is important to aspirate the 80% ethanol slowly and thoroughly to avoid any droplets remaining on the tube walls. If a small amount of liquid remains on the tube walls, briefly centrifuge the tube, place it back on the magnetic rack, and after thorough magnetic absorption, aspirate the liquid from the bottom of the tube using a small-range pipette. Allow to air dry with the lid open for 3-5 minutes until the magnetic beads are no longer reflective and have no signs of cracking (i.e., slight cracks in the bead cluster are observed). Add 32.5 μL of Buffer EB, close the tube cap, remove the PCR tube from the magnetic rack, vortex to mix, let stand at room temperature for 2 minutes, and briefly centrifuge to collect the solution at the bottom of the tube. Place the PCR tube on the magnetic rack and let stand for about 2 minutes until the magnetic attraction is sufficient and the liquid is clear. Aspirate 30 μL of the liquid into a new 1.5 mL centrifuge tube, avoiding the magnetic beads. This will obtain the purified product.

[0046] 1.5 Library construction 1.5.1 Fragmentation and end repair Remove the host-free Q-Buffer reagent, thaw, and spin down briefly. Add 120 μL of Buffer EB and vortex to mix thoroughly to prepare the Q-Buffer diluent. Place on ice until ready to use. Next, prepare the fragmentation and end-repair system as shown in Table 5. Once prepared, pipette up and down 10 times to mix thoroughly. Briefly centrifuge to collect the mixture at the bottom of the tube and place on ice until ready to use. Finally, set up the PCR reaction program as shown in Table 6. Once set up, incubate the prepared fragmentation and end-repair system in a PCR instrument. After incubation, obtain the fragmentation and end-repair products and place on ice until ready to use.

[0047] Table 5 Fragmentation and end-repair system Note: The DNA input amount in the table is ≤5 ng, and the input volume is ≤30 μL. The reagent configuration in this step needs to be double-checked by two people, and the reagent configuration can be carried out only after confirmation.

[0048] Table 6 Reaction procedure Note: This step requires double-checking of the PCR procedure. After confirmation, the PCR reaction can be carried out.

[0049] 1.5.2 Adapter ligation (Using the Universal Sequencing Reaction Preparation Kit, REF (Cat. No.): 2102, purchased from Tianjin Golden Key Medical Technology Co., Ltd.) First, prepare the adapter ligation system as shown in Table 7. After preparation, pipette up and down to mix 10 times. Briefly centrifuge to collect the mixture at the bottom of the tube and place it on ice until ready to use. Next, set up the PCR reaction program as shown in Table 8. After setting up, place the prepared adapter ligation system in a PCR instrument and incubate. After incubation, obtain the adapter ligation product and place it on ice until ready to use.

[0050] Table 7 Connector connection system Note: The UDB Adapter stock solution in the table is 10 μM and needs to be diluted before use. During the experiment, the reagents must be double-checked by two people, and only after confirmation can the reagents be prepared.

[0051] Table 8 Reaction procedure Note: This step requires double-checking of the PCR procedure. After confirmation, the PCR reaction can be carried out.

[0052] 1.5.3 Purification of adapter-ligated products To 100 μL of the adapter-ligated product obtained above, add 60 μL of DNA purification magnetic beads (stored in a 4°C refrigerator, take out 30 minutes in advance to equilibrate to room temperature, and vortex before use). Mix thoroughly by pipetting 10 times. Incubate at room temperature for 5 minutes, and briefly centrifuge to collect the mixture at the bottom of the tube. Next, place the PCR tube on a magnetic rack and let it sit for approximately 3-5 minutes. Once the liquid in the tube is clear, carefully aspirate and discard. Add 200 μL of 80% ethanol (freshly prepared immediately before use), let it sit for 30 seconds, and then carefully aspirate and discard the liquid. It is important to aspirate the 80% ethanol slowly and thoroughly to avoid any droplets remaining on the tube walls. If a small amount of liquid remains on the tube walls, briefly centrifuge the tube, place it back on the magnetic rack, and after thorough magnetic aspiration, aspirate the liquid from the bottom of the tube using a small-range pipette. Allow to air dry with the lid open for 3-5 minutes until the magnetic beads are no longer reflective and have no signs of cracking (i.e., slight cracks in the bead cluster are observed). Then, add 23 μL of Buffer EB, close the tube cap, remove the PCR tube from the magnetic rack, vortex to mix, let it stand at room temperature for 2 minutes, and briefly centrifuge to collect the solution at the bottom of the tube. Finally, place the PCR tube on the magnetic rack and let it stand for 2 minutes until it is fully magnetically attracted and the liquid is clear. Aspirate 21 μL of the liquid into a new PCR tube, avoiding the magnetic beads, to obtain the purified adapter ligation product.

[0053] 1.5.4 Library PCR Amplification First, prepare the library PCR reaction system as shown in Table 9. Use a pipette to gently pipette or tap the tube to mix (do not oscillate). Briefly centrifuge to collect the reaction solution at the bottom of the tube and place on ice until ready to use. Then, set up the PCR reaction program as shown in Table 10. After setup, place the prepared PCR reaction system in a thermal cycler for amplification. After amplification, obtain the library PCR product and place it on ice until ready to use.

[0054] Table 9 Library PCR amplification reaction system Note: 1) The amplification mix is ​​derived from the components in the Universal Sequencing Reaction Preparation Kit (REF: 2102, purchased from Tianjin Jinchi Medical Technology Co., Ltd.); 2) The UDB-XX PCR primer stock solution is 20 μM and needs to be diluted to 10 μM using the components in the MGIEasy Dual-Ended Individually Indexed Primer Adapter Kit B (Cat. No. 1000022802); 3) The reagent configuration in this step must be double-checked by two people and can only be continued after confirmation.

[0055] Table 10 PCR reaction program Note: This step requires double-checking of the PCR procedure. After confirmation, the PCR reaction can be carried out.

[0056] 1.5.5 Post-PCR Library Purification First, pipette 45 μL of DNA purification magnetic beads into 50 μL of PCR product. Vortex or pipette 10 times to mix thoroughly. Incubate at room temperature for 5 minutes, then briefly centrifuge to collect the mixture at the bottom of the tube. Add 200 μL of 80% ethanol (freshly prepared immediately), let it sit for 30 seconds, and carefully aspirate to remove all liquid. It is important to aspirate the 80% ethanol slowly and thoroughly to avoid any droplets remaining on the tube walls. If a small amount of liquid remains on the tube walls, briefly centrifuge the tube, place it back on the magnetic stand, and after thorough magnetic absorption, aspirate the liquid from the bottom of the tube using a small-range pipette. Allow to air dry with the lid open for 3-5 minutes until the magnetic beads are no longer reflective and have only slight cracks (i.e., slight cracks in the bead cluster are observed). Then, add 32.5 μL of Buffer EB, close the cap, remove the PCR tube from the magnetic stand, vortex to mix thoroughly, let it sit at room temperature for 2 minutes, and briefly centrifuge to collect the solution at the bottom of the tube. Finally, place the PCR tube on a magnetic stand and let it stand for about 2 minutes until it is fully magnetically attracted and the liquid is clear. Pipette out 30 μL of liquid into a new 1.5 mL centrifuge tube. Do not aspirate the magnetic beads to obtain the purified library.

[0057] Library concentration was determined using a double-stranded DNA (ds-DNA) concentration assay kit (fluorescence method) and a Qubit 4.0 fluorometer. Library concentration quality control standards are shown in Table 11.

[0058] Table 11 Library concentration quality control standards Note: The library concentration >1 ng / μL is considered qualified. If it fails, the library needs to be rebuilt. If the rebuilt library still fails, it needs to be fed back to the clinic for re-sending samples.

[0059] 1.6 Library Pooling Fill in the basic information of the libraries to be pooled in the pooling table (i.e., pooling table). Calculate the sample volume and theoretical concentration of the pooling solution for each library based on the library concentration and the required data volume. Arrange the libraries in the order of pooling and pipette the corresponding volume of each library into a 1.5 mL EP tube. Vortex to mix thoroughly and briefly centrifuge to collect the solution at the bottom of the tube. Measure the library concentration using the double-stranded DNA (ds-DNA) concentration assay kit (fluorimetric) and the Qubit 4.0 fluorometer. Enter the result in the "Measured Pooling Concentration" section of the pooling table. Compare the measured pooling concentration to the theoretical pooling concentration, and ensure the difference between the two values ​​is within 15%. If the difference is outside this range, repeat the Qubit assay.

[0060] Note: Try to control the aspiration volume of a single library within 0.5-4.5 μL to ensure that the library sampling volume is as accurate as possible and does not exceed the total volume of the library.

[0061] 1.7 DNB preparation 1.7.1 Reagent Preparation Remove the library, TE buffer, buffer MB, enzyme MA, enzyme MB, and DNB termination buffer (all from the Universal Sequencing Reaction Kit (Sequencing Method) DNB Preparation and Sequencing Kit, Cat. No.: 2010-02, Tianjin Golden Key Medical Technology Co., Ltd.), place on ice for approximately 0.5 h. After thawing, vortex and mix for 5 s, centrifuge briefly, and place on ice for later use.

[0062] 1.7.2 Preparation of DNB System 1 Prepare the reaction system on ice in a 0.2 mL eight-tube strip or PCR tube according to the system in Table 12 below. Vortex and mix thoroughly. Centrifuge briefly to collect the solution at the bottom of the tube. Place the tube in a PCR instrument and follow the protocol in Table 13 below to obtain DNB System 1.

[0063] Table 12 DNB reaction system Table 13 Reaction procedure 1.7.3 Preparation of DNB System 2 Remove Enzyme MB and place it on ice. Centrifuge briefly to collect the solution at the bottom of the tube, and place it on ice until ready for use. After the PCR reaction is complete, remove the PCR tube and centrifuge briefly to collect the solution at the bottom of the tube. Add the components listed in Table 14 below while still on ice. Vortex to mix thoroughly, centrifuge briefly to collect the solution at the bottom of the tube, and place it in a PCR instrument and perform the reaction according to the protocol listed in Table 15 below.

[0064] Table 14 DNB reaction system Table 15 Reaction procedure Note: The thermal cover of the PCR instrument needs to be preheated in advance to ensure that the thermal cover is at the working temperature during the DNB reaction. The thermal cover temperature is recommended to be set to 35°C, or as close to 35°C as possible.

[0065] Immediately after the reaction is completed, place it in an ice box, slowly add 20 μL of DNB termination buffer (about 1 drop) with a wide-bore pipette tip, adjust the pipette to 100 μL, slowly suck all the liquid with a wide-bore pipette tip, and then drop by drop into the mixed liquid in the pipette tip until the liquid is completely drained. Repeat 5-8 times, and store at 4°C for standby use (within 24 hours).

[0066] The concentration is determined using a single-stranded DNA (ssDNA) concentration determination kit (fluorescence method) and a Qubit 4.0 fluorometer. Concentration > 8 ng / μL is considered qualified, and if it does not meet the requirements, the DNB needs to be prepared again.

[0067] Note: a) Because DNB is sticky, it is recommended to take 2 μL for detection. If the number of samples is large, it is recommended to batch quantification to avoid fluorescence quenching, which may cause inaccurate quantification of DNB concentration; b) If the concentration exceeds 40 ng / μL, it needs to be diluted to about 20 ng / μL with DNB loading buffer I before use.

[0068] 1.8 Sequencing on the machine 1.8.1 Reagent preparation Take the slide out of the refrigerator, take the slide out of the packaging box but do not open the vacuum packaging bag, and place the slide at room temperature for at least 60 min (not more than 24 h). Open the vacuum packaging bag of the slide before use, start DNB loading, and the loading system is shown in Table 16. Mix the DNB loading system slowly with a wide-bore pipette tip for 5-8 times.

[0069] Table 16 DNB loading system Note: In the table: a) DNB loading buffer II is from the sequencing reaction general kit (REF: 2010-02; purchased from Tianjin Jinkey Medical Technology Co., Ltd.); b) If the slide is taken out of the refrigerator and has been placed at room temperature for more than 24 h, and the vacuum packaging bag is intact, it can be stored at 2-8°C, but the environment change between 2-8°C and room temperature should not exceed 3 times; c) The vacuum packaging bag should not be opened immediately and can be stored at room temperature for 24 h. If it is more than 24 h, it is not recommended to use it.

[0070] Thaw the reagents at room temperature for 3-4 hours and place them in a 4°C refrigerator until ready to use. The chip should equilibrate at room temperature for 30 minutes. Before use, mix the reagents by inverting them three times. Then, place the reagent reservoir directly in front of you and shake it back and forth 10-20 times. Open the reagent reservoir cover and wipe away any condensed water with a lint-free tissue. Remove dNTP Mix III and dNTP Mix II one hour in advance, thaw them at room temperature, and place them on ice or at 4°C until ready to use. Before adding samples, vortex and mix thoroughly for 5 seconds and briefly centrifuge before use. Remove the DNA polymerase mix and place them on ice or at 4°C until ready to use. Invert and mix thoroughly 4-6 times before adding samples. Using a clean 1 mL pipette tip, gently poke a sample well approximately 0.5 cm in diameter at the edge of wells 1 and 2. Using a pipette with the appropriate volume, add the corresponding reagents to the corresponding wells according to the volumes shown in Table 17 below. Then, place the reagent tank horizontally on the table, hold both sides with both hands, and shake it clockwise 10-20 times, then counterclockwise 10-20 times to ensure that the reagents are fully mixed. At this point, the preparation work for the sequencing reagent tank before loading is complete.

[0071] Table 17 Reagent loading volume Finally, sequencing was performed on the computer. The sequencing platform used was PanGenomics 3000, and the configured server parameters are shown in Table 1.

[0072] 1.9 Results and Analysis 1) Data quality is shown in Table 18 below: Table 18 Data quality of two strains 2) The results of local MLST typing tests are shown in Tables 19 and Figure 2-3 As shown: Table 19 Typing test results From Table 19 and Figure 2-3 As can be seen, the typing results of the two strains tested by the above method are: NJGLYY4758: type 39; NJGLYY2254: type 987. The total testing time is: 18 hours.

[0073] Example 3: Whole genome sequencing of Escherichia coli The two E. coli strains in Example 2 were sequenced and typed using whole genome sequencing (performed by Novogene), where: 1) The whole genome sequencing information of the strain is shown in Tables 20 and Figure 4-5 As shown: Table 20 Whole genome sequencing information 2) The final sequencing results are shown in Table 21 and Figure 6-7

[0074] Table 21 MLST prediction based on whole genome sequencing data of strains From the above results, it can be seen that the typing detection results of the two strains of Escherichia coli obtained by whole genome sequencing are: NJGLYY4758 belongs to type 39; NJGLYY2254 belongs to type 987. The results are consistent with the test results in Example 2. It can be seen that the detection method described in Example 2 has the advantages of high accuracy and short sequencing time.

[0075] The above is only a preferred embodiment of the present application, and does not limit the present application in any form. Although the present application has been disclosed as above with a preferred embodiment, it is not intended to limit the present application. Any person skilled in the art can make some changes or modifications to the above disclosed methods and technical contents without departing from the scope of the technical solution of the present application, and any simple modification, equivalent change and modification of the above embodiments made according to the technical essence of the present application still belong to the scope of the technical solution of the present application.​

Claims

1. A primer set for MLST typing of Escherichia coli based on a hospital-based microbial mNGS platform, characterized in that: The primers include a primer pair for amplifying the dinB gene, a primer pair for amplifying the icdA gene, a primer pair for amplifying the pabB gene, a primer pair for amplifying the polB gene, a primer pair for amplifying the putP gene, a primer pair for amplifying the trpB gene, a primer pair for amplifying the trpA gene, and a primer pair for amplifying the uidA gene; The amplification dinB The primer pair for the gene includes a forward primer Ecoli1_dinB-F having a nucleotide sequence as shown in SEQ ID NO: 1, and a reverse primer Ecoli1_dinB-R having a nucleotide sequence as shown in SEQ ID NO: 2; The amplification icdA The primer pair for the gene includes a forward primer Ecoli2_icdA-F having a nucleotide sequence as shown in SEQ ID NO: 3, and a reverse primer Ecoli2_icdA-R having a nucleotide sequence as shown in SEQ ID NO: 4; The amplification pabB The primer pair for the gene includes a forward primer Ecoli3_pabB-F having a nucleotide sequence as shown in SEQ ID NO: 5, and a reverse primer Ecoli3_pabB-R having a nucleotide sequence as shown in SEQ ID NO: 6; The amplification polB The primer pair for the gene includes a forward primer Ecoli4_polB-F having a nucleotide sequence as shown in SEQ ID NO: 7, and a reverse primer Ecoli4_polB-R having a nucleotide sequence as shown in SEQ ID NO: 8; The amplification putP The primer pair for the gene includes a forward primer Ecoli5_putP-F having a nucleotide sequence as shown in SEQ ID NO: 9, and a reverse primer Ecoli5_putP-R having a nucleotide sequence as shown in SEQ ID NO: 10; The amplification trpB The primer pair for the gene includes a forward primer Ecoli6_trpB-F having a nucleotide sequence as shown in SEQ ID NO: 11, and a reverse primer Ecoli6_trpB-R having a nucleotide sequence as shown in SEQ ID NO: 12; The amplification trpA The primer pair for the gene includes a forward primer Ecoli7_trpA-F having a nucleotide sequence as shown in SEQ ID NO: 13, and a reverse primer Ecoli7_trpA-R having a nucleotide sequence as shown in SEQ ID NO: 14; and The amplification uidA The primer pair for the gene includes a forward primer Ecoli8_uidA-F whose nucleotide sequence is shown in SEQ ID NO: 15, and a reverse primer Ecoli8_uidA-R whose nucleotide sequence is shown in SEQ ID NO:

16.

2. A kit for MLST typing of Escherichia coli based on a hospital-based microbial mNGS platform, characterized in that: The kit comprises the primer set according to claim 1, and the concentration of each primer is independently 10 μM.

3. The kit according to claim 2, wherein The kit also includes 2X TaqMan FastqPCR Master Mix.

4. Use of the primer set according to claim 1 or the kit according to claim 2 or 3 in constructing an mNGS sequencing library for Escherichia coli MLST typing.

5. A method for constructing an mNGS sequencing library for MLST typing of Escherichia coli, characterized in that: The following steps are involved: 1) Extract DNA from the sample to be tested; 2) performing a qPCR amplification reaction on the DNA of the sample to be tested extracted in step 1) using the kit according to claim 2 or 3; 3) Add mNGS sequencing adapters to both ends of the amplification reaction product of step 2) to obtain an mNGS sequencing library.

6. The method according to claim 5, characterized in that In step 1), DNA is extracted from the sample to be tested using a bacterial genomic DNA extraction kit.

7. The method according to claim 5, characterized in that In step 2), the reaction conditions for qPCR amplification of the test sample DNA are as follows: 94°C for 2 min, cycle number 1; 94°C for 30 s, cycle number 35; 50°C for 1 min; 72°C for 30 s; 72°C for 5 min, cycle number 1; 4°C forever.

8. The method according to claim 5, characterized in that In step 2), the reaction system for qPCR amplification of the sample DNA to be tested is composed of: 。 9. The method according to claim 5, characterized in that The instrument used for mNGS sequencing was PanGenomics 2000, and the detection server used was CentOS Linux release 7.9.2009 (Core).

10. Use of the primer set according to claim 1, or the kit according to claim 2 or 3, or the method according to any one of claims 5 to 9 in preparing a reagent for detecting MLST typing of Escherichia coli.

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