Primer group and method for Pseudomonas aeruginosa MLST typing based on hospital localized microbe mNGS platform

Abstract

The invention belongs to the technical field of microbiological detection, and particularly relates to a pseudomonas aeruginosa MLST typing primer group and method based on a hospital localized microbiological mNGS platform. The primer group comprises a primer pair for amplifying an aroE gene, a trpE gene, an acsA gene, a ppsA gene, a nuoD gene, a guaA gene and a mutL gene, and corresponding nucleotide sequences of the primer pair are as shown in SEQ ID NO: 1-14. 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, the primer group for amplifying the MLST housekeeping gene of the pseudomonas aeruginosa is redesigned, and the length and the position of an amplicon are optimized, so that the amplicon adapts to the sequencing working principle of the existing next-generation sequencing platform in the hospital. 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 Pseudomonas aeruginosa 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, and finally determine the ST type. 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. On another level, if we start from the perspective of scientific research, there is also a method of performing cgMLST analysis through whole genome sequencing. 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 work.

[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 Pseudomonas aeruginosa based on a hospital-based microbial mNGS platform, so as to achieve rapid and accurate typing detection of Pseudomonas aeruginosa.

[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 Pseudomonas aeruginosa based on a hospital-based localized microbial mNGS platform, comprising a primer pair for amplifying the aroE gene, a primer pair for amplifying the trpE gene, a primer pair for amplifying the acsA gene, a primer pair for amplifying the ppsA gene, a primer pair for amplifying the nuoD gene, a primer pair for amplifying the guaA gene, and a primer pair for amplifying the mutL gene; The amplification aroE The primer pair for the gene includes a forward primer PA_1.aroE-F having a nucleotide sequence as shown in SEQ ID NO: 1, and a reverse primer PA_1.aroE-R having a nucleotide sequence as shown in SEQ ID NO: 2; The amplification trpE The primer pair for the gene includes a forward primer PA_2.trpE-F having a nucleotide sequence as shown in SEQ ID NO: 3, and a reverse primer PA_2.trpE-R having a nucleotide sequence as shown in SEQ ID NO: 4; The amplification acsA The primer pair for the gene includes a forward primer PA_3.acsA-F having a nucleotide sequence as shown in SEQ ID NO: 5, and a reverse primer PA_3.acsA-R having a nucleotide sequence as shown in SEQ ID NO: 6; The amplification ppsA The primer pair for the gene includes a forward primer PA_4.ppsA-F having a nucleotide sequence as shown in SEQ ID NO: 7, and a reverse primer PA_4.ppsA-R having a nucleotide sequence as shown in SEQ ID NO: 8; The amplification nuoD The primer pair for the gene includes a forward primer PA_5.nuoD-F having a nucleotide sequence as shown in SEQ ID NO: 9, and a reverse primer PA_5.nuoD-R having a nucleotide sequence as shown in SEQ ID NO: 10; The amplification guaA The primer pair for the gene includes a forward primer PA_6.guaA-F having a nucleotide sequence as shown in SEQ ID NO: 11 and a reverse primer PA_6.guaA-R having a nucleotide sequence as shown in SEQ ID NO: 12; and The amplification mutL The primer pair for the gene includes a forward primer PA_7.mutL-F having a nucleotide sequence as shown in SEQ ID NO: 13, and a reverse primer PA_7.mutL-R having a nucleotide sequence as shown in SEQ ID NO: 14.

[0011] The second aspect of the present application provides a kit for P. aeruginosa MLST typing based on a hospital-localized microbial mNGS platform, the kit comprising the primer set as previously described.

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

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

[0014] The third aspect of the present application provides a use of the primer set or the kit as previously described in P. aeruginosa MLST typing traceability of P. aeruginosa mNGS sequencing library construction.

[0015] The fourth aspect of the present application provides a method for P. aeruginosa MLST typing traceability of mNGS sequencing library construction, comprising the following steps: 1) extracting DNA from the sample to be tested; 2) performing qPCR amplification reaction on the DNA from the sample to be tested extracted in step 1) using the kit as previously described; 3) adding 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 application, 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 application, in step 2), the reaction conditions for performing qPCR amplification reaction on the DNA from the sample to be tested are as follows: 94℃ for 2 min, cycle number 1; 94℃ for 30 s, cycle number 35; 50℃ for 1 min; 72℃ for 30 s; 72℃ for 5 min, cycle number 1; 4℃ forever.

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

[0019] The fifth aspect of the present application provides a use of the primer set, or the kit, or the method as described above in the preparation of a reagent for detecting Pseudomonas aeruginosa MLST typing traceability.

[0020] By the above technical solution, the present application has at least the following advantages: The present application re-designs the primer for amplifying the housekeeping gene of Pseudomonas aeruginosa MLST according to the technical characteristics of the short sequencing read length and single-end sequencing of the in-hospital second-generation sequencing platform, improves the multi-locus sequence analysis (MLST) technology by lengthening the amplicon length, and makes it suitable for the existing in-hospital second-generation sequencing platform. The product amplified by the primer set of the present application is used for library construction, and a specific second-generation sequencing platform is used for sequencing, which can realize rapid and accurate homology analysis of Pseudomonas aeruginosa, and assist in the prevention and control of in-hospital infection. The primer set, kit or method of the present application can quickly (within 24 hours) and accurately determine the genetic relationship between in-hospital pathogenic bacteria, significantly improve the timeliness and scientificity of infection prevention and control, reduce the incidence of in-hospital infection, reduce the hospitalization time and medical expenses of patients, and improve the safety of patients and the reputation of hospitals in the long term, which has a broad clinical application prospect.

[0021] The present application optimizes the primer design and sequencing process, so that while daily clinical routine mNGS detection is carried out, bacterial MLST typing detection can be carried out using the remaining positions of the chip. This not only improves the detection efficiency, but also reduces the cost, while maintaining high precision and high timeliness. It can provide a more efficient and economical bacterial typing solution for hospitals and centers for disease control.

[0022] The primer set, kit and corresponding sequencing method of the present application can also promote regional in-hospital infection prevention and control cooperation, improve regional public health level, and lay a foundation for subsequent technology upgrading and wider clinical application, which has important academic value and social significance for improving medical quality and promoting public health development.

[0023] The above description is only a summary of the technical solutions of the present application. In order to more clearly understand the technical means of the present application and can be implemented according to the content of the specification, the following will be described in detail with the preferred embodiments of the present application. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 The colony morphology of two strains of Pseudomonas aeruginosa NJGLYY_19-49 and NJGLYY_21-54 is shown; wherein the upper graph is the colony morphology after 24 hours of culture at 35℃ in blood agar medium with 5% CO2; the lower graph is the colony morphology after 24 hours of culture at 35℃ in MH agar medium; Figure 2 The typing detection result graph of strain NJGLYY_19-49 in Example 2 is shown; Figure 3 A graph showing the typing detection results of strain NJGLYY_21-54 in Example 2 is shown; Figure 4 A graph showing the whole genome sequencing quality analysis of strain NJGLYY_19-49 in Example 3 is shown; Figure 5 A graph showing the whole genome sequencing quality analysis of strain NJGLYY_21-54 in Example 3 is shown; Figure 6 A graph showing the whole genome sequencing circle of strain NJGLYY_19-49 in Example 3 is shown; Figure 7 A graph showing the whole genome sequencing circle of strain NJGLYY_21-54 in Example 3 is shown; Figure 8 A graph showing the whole genome sequencing analysis detection results of strain NJGLYY_19-49 in Example 3 is shown; Figure 9 A graph showing the whole genome sequencing analysis detection results of strain NJGLYY_21-54 in Example 3 is shown. DETAILED DESCRIPTION

[0025] In order to make the technical means, creative features, purposes and effects realized by the present application easy to understand, the technical solutions in the embodiments of the present application will be described clearly and completely below in conjunction with the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative labor fall within the scope of protection of the present application.

[0026] The traditional MLST technology is based on first-generation sequencing, and the positions of the primers and the lengths of the amplicons are fully considered in view of the technical characteristics of the first-generation sequencing. However, the primers are redesigned and the lengths of the amplicons are adjusted according to the characteristics of the second-generation sequencing platform in the present application.

[0027] Therefore, in one embodiment, the present application provides a primer set for Pseudomonas aeruginosa MLST typing based on a hospital-localized microbial mNGS platform, which comprises a primer pair for amplifying the aroE gene, a primer pair for amplifying the trpE gene, a primer pair for amplifying the acsA gene, a primer pair for amplifying the ppsA gene, a primer pair for amplifying the nuoD gene, a primer pair for amplifying the guaA gene, and a primer pair for amplifying the mutL gene. The primer pair for amplifying the aroE gene comprises a forward primer PA_1.aroE-F having a nucleotide sequence as set forth in SEQ ID NO: 1, and a reverse primer PA_1.aroE-R having a nucleotide sequence as set forth in SEQ ID NO: 2; The primer pair for amplifying the trpE gene comprises a forward primer PA_2.trpE-F having a nucleotide sequence as set forth in SEQ ID NO: 3, and a reverse primer PA_2.trpE-R having a nucleotide sequence as set forth in SEQ ID NO: 4; The primer pair for amplifying the acsA gene comprises a forward primer PA_3.acsA-F having a nucleotide sequence as set forth in SEQ ID NO: 5, and a reverse primer PA_3.acsA-R having a nucleotide sequence as set forth in SEQ ID NO: 6; The primer pair for amplifying the ppsA gene comprises a forward primer PA_4.ppsA-F having a nucleotide sequence as set forth in SEQ ID NO: 7, and a reverse primer PA_4.ppsA-R having a nucleotide sequence as set forth in SEQ ID NO: 8; The primer pair for amplifying the nuoD gene comprises a forward primer PA_5.nuoD-F having a nucleotide sequence as set forth in SEQ ID NO: 9, and a reverse primer PA_5.nuoD-R having a nucleotide sequence as set forth in SEQ ID NO: 10; The primer pair for amplifying the guaA gene comprises a forward primer PA_6.guaA-F having a nucleotide sequence as set forth in SEQ ID NO: 11, and a reverse primer PA_6.guaA-R having a nucleotide sequence as set forth in SEQ ID NO: 12; and The primer pair for amplifying the mutL gene comprises a forward primer PA_7.mutL-F having a nucleotide sequence as set forth in SEQ ID NO: 13, and a reverse primer PA_7.mutL-R having a nucleotide sequence as set forth in SEQ ID NO: 14. Exemplarily, the specific sequence information of the above primer set is shown in the following table: The workload can be greatly reduced by constructing multiplex amplification primers. When designing multiplex amplification primers, cross-reaction or non-specific amplification between primers should be avoided as much as possible. In addition, the amplification conditions such as primer concentration and annealing temperature need to be optimized to ensure the consistency of the target gene amplification efficiency. Overall, through the two aspects of bioinformatics prediction and experimental optimization, constructing a set of efficient and stable multiplex amplification primer combination to provide qualified DNA library for sequencing is another key technical innovation of the present application.

[0028] In another embodiment, a kit for MLST typing and tracing of Pseudomonas aeruginosa is provided, wherein the kit comprises the above-mentioned primer set.

[0029] Preferably, the kit further comprises 2X TaqMan Fast qPCR Master Mix.

[0030] In a further optimized example, the concentration of each primer in the kit was independently 10 µM.

[0031] In yet another embodiment, there is provided a use of the aforementioned primer set or kit in constructing a mNGS sequencing library for Pseudomonas aeruginosa MLST typing and tracing.

[0032] By applying the primer set and kit to construct mNGS sequencing libraries for Pseudomonas aeruginosa MLST typing and tracing, the kit 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.

[0033] In another embodiment, a method for constructing an mNGS sequencing library for MLST typing and tracing of Pseudomonas aeruginosa 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.

[0034] In step 1), DNA is extracted from the sample to be tested using a bacterial genomic DNA extraction kit.

[0035] 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.

[0036] 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.

[0037] The following embodiments relate to and refer to: 1. mNGS detection platform for MLST typing and traceability of Pseudomonas aeruginosa: The instrument used is PanGenomics 2000, and the configured server is shown in Table 1.

[0038] 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 Pseudomonas aeruginosa are: nuoD、trpE、guaA、 acsA, ppsA, aroE, mutL (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.

[0039] 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 " Pseudomona aeruginosa "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. aroE Primer pairs for gene amplification trpE Primer pairs for gene amplification acsA Primer pairs for gene amplification ppsA Primer pairs for gene amplification nuoD Primer pairs for gene amplification guaA Primer pairs and amplification of genes mutL The primer pairs for the genes are shown in SEQ ID NOs: 1 to 14. 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 Pseudomonas aeruginosa In this example, two strains of Pseudomonas aeruginosa isolated from the sputum of the subjects were used as test samples, and the primer set determined in Example 1 was used to perform MLST typing detection on the test samples, as follows: 1.1 Strain source and information The strains used in this example were isolated from the sputum of patients visiting Nanjing Drum Tower Hospital. Their specific information is shown in Table 3: Table 3 Pseudomonas aeruginosa strain information like Figure 1 Shown are the colony morphologies of two strains of Pseudomonas aeruginosa, NJGLYY_19-49 and NJGLYY_21-54; the upper figure is the colony morphology after culture on blood agar medium at 35°C and 5% CO2 for 24 hours; the lower figure is the colony morphology after culture on MH agar medium at 35°C for 24 hours.

[0040] 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.

[0041] 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.

[0042] 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.

[0043] The PCR amplification reaction was performed according to the above reaction system and procedure to obtain the PCR reaction product.

[0044] 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.

[0045] 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.

[0046] 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.

[0047] Table 6 Reaction procedure Note: This step requires double-checking of the PCR procedure. After confirmation, the PCR reaction can be carried out.

[0048] 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.

[0049] 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.

[0050] Table 8 Reaction procedure Note: This step requires double-checking of the PCR procedure. After confirmation, the PCR reaction can be carried out.

[0051] 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.

[0052] 1.5.4 Library PCR Amplification First, prepare the library PCR reaction system as shown in Table 9, mix gently with a pipette or tap the tube wall, do not shake, and centrifuge briefly to collect the reaction solution at the bottom of the tube, and place it on ice. Then set the PCR reaction program as shown in Table 10, after setting, place the prepared PCR reaction system in the PCR instrument for amplification, and obtain the library PCR amplification product after amplification, and place it on ice.

[0053] Table 9 Library PCR amplification reaction system Note: 1) The amplification mixture is from the components in the sequencing reaction preparation universal kit (REF (article number): 2102, purchased from Tianjin Jinkey Medical Technology Co., Ltd.); 2) The UDB-XX PCR primer stock solution is 20 μM, which needs to be diluted to 10 μM, and the components from the MGIEasy double-end independent tag primer linker kit B (article number: 1000022802); 3) The reagent configuration of this step needs to be double-checked by two people, and the reagent configuration can be performed after confirming that there is no error.

[0054] Table 10 PCR reaction program Note: This step needs to double-check the PCR program, and the PCR reaction can be performed after confirming that there is no error.

[0055] 1.5.5 Library purification after PCR First, take 45 μL DNA purification magnetic beads into 50 μL PCR reaction product, vortex or use a pipette to beat 10 times for thorough mixing, incubate at room temperature for 5 min, and centrifuge briefly to collect the mixture at the bottom of the tube. Add 200 μL of 80% ethanol (freshly prepared), stand for 30 s, and carefully aspirate all the liquid; it should be noted that the 80% ethanol should be aspirated slowly, and the liquid droplets should be aspirated as much as possible to avoid residual liquid on the wall of the tube; if there is a small amount of liquid remaining on the wall of the tube, the centrifuge tube can be centrifuged briefly, placed on the magnetic stand for full magnetic attraction, and the liquid at the bottom of the tube can be aspirated with a small volume pipette. Open the cap and dry for 3-5 min until the magnetic beads have no reflection and are slightly cracked (i.e., a slight crack is observed in the magnetic bead group). Then add 32.5 μL Buffer EB, remove the PCR tube from the magnetic stand, vortex to mix, and incubate at room temperature for 2 min. Centrifuge briefly to collect the solution at the bottom of the tube. Finally, place the PCR tube on the magnetic stand, stand for about 2 min until the liquid is fully magnetically attracted and clarified, and aspirate 30 μL of liquid into a new 1.5 mL centrifuge tube. Do not aspirate the magnetic beads to obtain the purified library.

[0056] The library concentration was determined using a double-stranded DNA (ds-DNA) concentration determination kit (fluorescence method) and a Qubit 4.0 fluorometer. The library concentration quality control standard is shown in Table 11.

[0057] Table 11 Library concentration quality control standard Note: The library concentration > 1 ng / μL is considered qualified, if not qualified, re-sequencing is required; if re-sequencing is still not qualified, feedback to the clinic for re-sampling is required.

[0058] 1.6 Library mixing The library information to be mixed is filled into the mixing operation table (i.e. pooling table), and the sampling volume of each library and the theoretical concentration of the pooling liquid are calculated according to the library concentration and the required data volume. The libraries are arranged in the mixing order, and the corresponding volume of each library is drawn into a 1.5 mL EP tube according to the calculated mixing volume, shaken and mixed, and the solution is collected at the bottom of the tube after short centrifugation. The double-stranded DNA (ds-DNA) concentration determination kit (fluorescence method) and Qubit 4.0 fluorometer are used to determine the library concentration, and the results are filled into the "pooling measured concentration" in the mixing operation table. The measured mixing concentration is compared with the theoretical mixing concentration, and the difference between the two is controlled within 15%. If the difference is not within this range, Qubit detection should be performed again.

[0059] Note: The sampling volume of each library is controlled within 0.5-4.5 μL as much as possible to ensure that the sampling volume of the library is as accurate as possible and does not exceed the total volume of the library.

[0060] 1.7 DNB preparation 1.7.1 Reagent preparation Take out the library, TE buffer, buffer MB, enzyme MA, enzyme MB and DNB termination buffer (all from the sequencing reaction general reagent kit (sequencing method) DNB preparation and sequencing reagent kit, item number: 2010-02, Tianjin Jinkey Medical Technology Co., Ltd.), and place them in an ice box for about 0.5 h. After melting, use a vortex shaker to shake and mix for 5 s, and centrifuge briefly and place on ice for standby.

[0061] 1.7.2 Preparation of DNB system 1 Use 0.2 mL eight-tube or PCR tube to prepare the reaction system as shown in Table 12 below on ice. Shake and mix with a vortex shaker, centrifuge briefly to collect the solution at the bottom of the tube, and place it in a PCR instrument for reaction according to the program shown in Table 13 below to obtain DNB system 1.

[0062] 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.

[0063] 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.

[0064] After the reaction is completed, immediately place it in an ice box and slowly add 20 µL of DNB stop buffer (about 1 drop) using a wide-mouth pipette tip. Adjust the pipette to 100 µL and slowly aspirate all the liquid with a wide-mouth pipette tip. Then add dropwise to mix until the liquid in the pipette tip is drained. Repeat 5-8 times. It can be stored at 4°C until used within 24 hours.

[0065] Measure the concentration using the single-stranded DNA (ssDNA) concentration assay kit (fluorescence) and a Qubit 4.0 fluorometer. A concentration >8 ng / µL is considered acceptable. If not, re-prepare DNB.

[0066] Note: a) Because DNB is relatively viscous, it is recommended to sample 2 μL for testing. For large samples, batch quantification is recommended to avoid inaccurate DNB concentration quantification due to fluorescence quenching. b) If the concentration exceeds 40 ng / µL, dilute to approximately 20 ng / µL with DNB Loading Buffer I before use.

[0067] 1.8 Sequencing 1.8.1 Reagent Preparation Remove the slides from the refrigerator and from the packaging, but leave them in the vacuum bag. Allow the slides to stand at room temperature for at least 60 minutes (no more than 24 hours). Open the vacuum bag again before use and begin DNB loading using the loading system shown in Table 16. Mix the DNB loading system gently 5-8 times with a wide-mouth pipette tip.

[0068] Table 16 DNB loading system Note: In the table: a) DNB loading buffer II from the sequencing reaction universal kit (REF: 2010-02; purchased from Tianjin Jinkey Medical Technology Co., Ltd.); b) If the slide cannot be used within 24 h after being taken out of the refrigerator and has been placed at room temperature, and the vacuum packaging bag is intact, it can be stored at 2-8°C again, but the environment switching between 2-8°C and room temperature should not exceed 3 times; c) The vacuum packaging bag cannot be used immediately after opening, and can be stored at room temperature and used within 24 h, and it is not recommended to use it if it exceeds 24 h.

[0069] Thaw the reagents at room temperature for 3-4 h, and store them in a 4°C refrigerator for standby, and the chip needs to be balanced at room temperature for 30 min; before use, the reagent needs to be inverted and mixed 3 times, then the reagent slot is placed in front, and shaken left and right 10-20 times. Open the reagent slot cover plate and wipe off the condensate with a dust-free paper. Take out dNTPs mixture III and dNTPs mixture II 1 h in advance, melt at room temperature, and store on ice or at 4°C for standby; before use, shake and mix for 5 s, and then use after a brief centrifugation. Take out the DNA polymerase mixture before use, and store it on ice or at 4°C for standby; before use, invert and mix 4-6 times. Use a clean 1 mL gun head to gently poke a sample hole with a diameter of about 0.5 cm at the edge of No. 1 and No. 2 holes. Take the corresponding range of pipettor, and add the corresponding reagent to the corresponding hole according to the volume shown in Table 17 below. Then place the reagent slot horizontally on the table, hold both sides with both hands, shake clockwise 10-20 times, and then counterclockwise 10-20 times to ensure thorough mixing of the reagents. At this time, the preparation of the sequencing reagent slot before machine is completed.

[0070] Table 17 Reagent sample amount Finally, the sequencing operation is carried out, and the sequencing platform used is Pan Genomics 3000, and the server parameters configured are shown in Table 1.

[0071] 1.9 Results and analysis 1) The data quality is shown in Table 18 below: Table 18 Data quality of two strains 2) The local MLST typing detection results are shown in Tables 19 and Figure 2-3 Table 19 Typing detection results From Tables 19 and Figure 2-3 ​As can be seen, the typing results of the two strains tested using the above method are: NJGLYY_19-49: type 258; NJGLYY_21-54: type 980. The total testing time is: 18 hours.

[0072] Example 3: Whole genome sequencing of Pseudomonas aeruginosa The two strains of Pseudomonas aeruginosa 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 whole genome sequencing diagrams of the two strains are as follows Figure 6-7 shown 3) The final sequencing results are shown in Table 21 and Figure 8-9 shown.

[0073] Table 21 MLST prediction based on whole genome sequencing data of strains The above results indicate that the typing results of the two Pseudomonas aeruginosa strains obtained by whole-genome sequencing are: NJGLYY_19-49 belongs to the ST258 type; NJGLYY_21-54 belongs to the ST980 type. This result is consistent with the test results in Example 2. Therefore, the detection method described in Example 2 of the present invention has the advantages of high accuracy and short sequencing time.

[0074] The above description is merely a preferred embodiment of the present invention and does not constitute any form of limitation to the present invention. Although the present invention has been disclosed as a preferred embodiment as above, it is not intended to limit the present invention. Any technician familiar with this profession can make slight changes or modifications to equivalent embodiments of the methods and technical contents disclosed above without departing from the scope of the technical solution of the present invention. However, any simple modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. A primer set for MLST typing of Pseudomonas aeruginosa based on a hospital-based microbial mNGS platform, characterized in that: The primers include a primer pair for amplifying the aroE gene, a primer pair for amplifying the trpE gene, a primer pair for amplifying the acsA gene, a primer pair for amplifying the ppsA gene, a primer pair for amplifying the nuoD gene, a primer pair for amplifying the guaA gene, and a primer pair for amplifying the mutL gene; The amplification aroE The primer pair for the gene includes a forward primer PA_1.aroE-F having a nucleotide sequence as shown in SEQ ID NO: 1, and a reverse primer PA_1.aroE-R having a nucleotide sequence as shown in SEQ ID NO: 2; The amplification trpE The primer pair for the gene includes a forward primer PA_2.trpE-F having a nucleotide sequence as shown in SEQ ID NO: 3, and a reverse primer PA_2.trpE-R having a nucleotide sequence as shown in SEQ ID NO: 4; The amplification acsA The primer pair for the gene includes a forward primer PA_3.acsA-F having a nucleotide sequence as shown in SEQ ID NO: 5, and a reverse primer PA_3.acsA-R having a nucleotide sequence as shown in SEQ ID NO: 6; The amplification ppsA The primer pair for the gene includes a forward primer PA_4.ppsA-F having a nucleotide sequence as shown in SEQ ID NO: 7, and a reverse primer PA_4.ppsA-R having a nucleotide sequence as shown in SEQ ID NO: 8; The amplification nuoD The primer pair for the gene includes a forward primer PA_5.nuoD-F having a nucleotide sequence as shown in SEQ ID NO: 9, and a reverse primer PA_5.nuoD-R having a nucleotide sequence as shown in SEQ ID NO: 10; The amplification guaA The primer pair for the gene includes a forward primer PA_6.guaA-F having a nucleotide sequence as shown in SEQ ID NO: 11 and a reverse primer PA_6.guaA-R having a nucleotide sequence as shown in SEQ ID NO: 12; and The amplification mutL The primer pair for the gene includes a forward primer PA_7.mutL-F having a nucleotide sequence as shown in SEQ ID NO: 13, and a reverse primer PA_7.mutL-R having a nucleotide sequence as shown in SEQ ID NO:

14.

2. A kit for MLST typing of Pseudomonas aeruginosa 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 a library for mNGS sequencing of Pseudomonas aeruginosa for MLST typing of Pseudomonas aeruginosa.

5. A mNGS sequencing library construction method for MLST typing of Pseudomonas aeruginosa, 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 Pseudomonas aeruginosa.

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