Primer group and method for staphylococcus aureus MLST typing based on hospital localized microorganism mNGS platform

Abstract

The invention belongs to the technical field of microbiological detection, and particularly relates to a staphylococcus aureus MLST typing primer group and method based on a hospital localized microbiological mNGS platform. The primer group comprises primer pairs for amplifying a yqiL gene, a pta gene, a tpi gene, a gmk gene, a glpF gene, an aroE gene and an arcC gene, and corresponding nucleotide sequences 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 invention redesigns a staphylococcus aureus MLST housekeeping gene amplification primer group, and by optimizing the length and position of amplicons, the amplicons are adaptive 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 application belongs to the technical field of microorganism detection, and particularly relates to a primer set and method for staphylococcus aureus MLST typing based on a hospital localized microorganism mNGS platform. BACKGROUND

[0002] Hospital infection, also known as hospital-acquired infection, refers to a disease caused by infection during treatment or care in a hospital. The hospital should actively take measures to prevent and control the occurrence of hospital infection to protect the health and safety of patients. Early detection and early treatment are key links. Only by identifying infection signs in time and taking effective measures quickly can the impact of infection on patients be minimized and further spread be prevented. In order to determine whether hospital infection occurs, bacteria need to be "typed" to determine whether the same type of bacteria occurs to judge whether a hospital infection event occurs, which provides a basis for subsequent isolation, tracing and other measures.

[0003] At present, there are various methods for microorganism typing, mainly including pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST) and whole genome sequencing (WGS) and the like. Among them, the MLST technology has the advantages of high precision and unified standard, and is a relatively common method at present. MLST is a technology for typing based on the nucleic acid sequences of multiple conservative gene sites, and these sequences are internationally agreed and known. MLST relies on the difference in bacterial conservative gene sequences for typing, so the process is divided into two steps of sequencing and database comparison. According to the type corresponding to each gene, it is mapped to a total "ST" type.

[0004] In actual work, by analyzing the sequences of multiple conservative gene sites, if the bacteria infected by multiple patients in the same ward are of the same ST type, it is suggested that there may be hospital infection or explosive spread. On the contrary, if the ST types of bacteria infected by different patients are quite different, it can be excluded that there is close spread between bacteria, so as to exclude hospital infection. Therefore, the typing detection of bacteria is an important basis for judging the trend of bacterial hospital dissemination or prevalence.

[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 according to the combination of each bacterial housekeeping gene, then send the amplification product to a sequencing company for detection, upload the data to an overseas website for query, and finally determine the ST type. The process has the following shortcomings: (1) Equipment and technical limitations: There are generally no related equipment and technology in hospitals and CDCs, and only third-party institutions can be sent for detection. The logistics time needs to be consumed during the sending process, which seriously affects the timeliness. (2) Result transmission and manual operation: After sequencing is completed, the test results are transmitted through the website or email, but the final splicing and comparison still need human operation, which is time-consuming and laborious and prone to errors. (3) Timeliness: The whole process of amplification, first-generation sequencing, determination of bacterial ST typing, and data arrangement needs at least 3 days to be reliable, but the second-generation sequencing for scientific research also has the problem of sample outsourcing sequencing, and the sequencing period is long~3 days, which cannot meet the actual needs of clinical infection prevention and control. (4) Data security: The determination and query of ST type need to upload data to an overseas website, which has the risk of domestic data leakage. From another perspective, if from the perspective of scientific research, there is also a method of cgMLST analysis through whole genome sequencing. This method produces a large amount of data, and the result takes 7~15 days, which is only suitable for retrospective research, and cannot provide timely guidance for clinical nosocomial infection work.

[0006] Therefore, at present, when carrying out bacterial MLST typing for nosocomial infection prevention and control work, although the first-generation sequencing technology has poor timeliness, complex operation and information leakage, it is still the main method selected at present.

[0007] In order to solve the diagnosis problem of patient's difficult infectious disease within 24 hours, some domestic first-class hospitals actively construct local second-generation sequencing platform, so as to carry out pathogenic microorganism second-generation sequencing detection service in the hospital (not outsourcing to the sequencing company). Although the purpose of constructing the local sequencing platform is to carry out pathogenic microorganism detection work, it also provides material basis and technical support for us to utilize the platform and carry out nosocomial infection bacterial homology analysis through technical innovation. SUMMARY

[0008] The purpose of the present application is to provide a primer set and method for Staphylococcus aureus MLST typing based on a hospital local microorganism mNGS platform, so as to realize rapid and accurate typing detection of Staphylococcus aureus.

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

[0010] The first aspect of the present application provides a primer set for Staphylococcus aureus MLST typing based on a hospital-localized microbial mNGS platform, comprising a primer pair for amplifying a yqiL gene, a primer pair for amplifying a pta gene, a primer pair for amplifying a tpi gene, a primer pair for amplifying a gmk gene, a primer pair for amplifying a glpF gene, a primer pair for amplifying an aroE gene, and a primer pair for amplifying an arcC gene. The primer pair for amplifying the yqiL gene comprises a forward primer SAU_1.yqiL-F with a nucleotide sequence as shown in SEQ ID NO: 1, and a reverse primer SAU_1.yqiL-R with a nucleotide sequence as shown in SEQ ID NO: 2. The primer pair for amplifying the pta gene comprises a forward primer SAU_2.pta-F with a nucleotide sequence as shown in SEQ ID NO: 3, and a reverse primer SAU_2.pta-R with a nucleotide sequence as shown in SEQ ID NO: 4. The primer pair for amplifying the tpi gene comprises a forward primer SAU_3.tpi-F with a nucleotide sequence as shown in SEQ ID NO: 5, and a reverse primer SAU_3.tpi-R with a nucleotide sequence as shown in SEQ ID NO: 6. The primer pair for amplifying the gmk gene comprises a forward primer SAU_4.gmk-F with a nucleotide sequence as shown in SEQ ID NO: 7, and a reverse primer SAU_4.gmk-R with a nucleotide sequence as shown in SEQ ID NO: 8. The primer pair for amplifying the glpF gene comprises a forward primer SAU_5.glpF-F with a nucleotide sequence as shown in SEQ ID NO: 9, and a reverse primer SAU_5.glpF-R with a nucleotide sequence as shown in SEQ ID NO: 10. The primer pair for amplifying the aroE gene comprises a forward primer SAU_6.aroE-F with a nucleotide sequence as shown in SEQ ID NO: 11, and a reverse primer SAU_6.aroE-R with a nucleotide sequence as shown in SEQ ID NO: 12. The primer pair for amplifying the arcC gene comprises a forward primer SAU_7.arcC-F with a nucleotide sequence as shown in SEQ ID NO: 13, and a reverse primer SAU_7.arcC-R with a nucleotide sequence as shown in SEQ ID NO: 14.

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

[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 described above in the library construction of mNGS sequencing for Staphylococcus aureus MLST typing.

[0015] The fourth aspect of the present application provides a method for library construction of mNGS sequencing for Staphylococcus aureus MLST typing, comprising the following steps: 1) extracting DNA from the sample to be tested; 2) performing qPCR amplification reaction on the DNA extracted from the sample to be tested in step 1) using the kit as described above; 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 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 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 Staphylococcus aureus MLST typing.

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

[0022] The primer set, kit and corresponding sequencing method of the present application can also promote regional nosocomial 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 the contents of the specification can be implemented as follows. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 The colony morphology of two strains of Staphylococcus aureus NJGLYY_12739 and NJGLYY_15701 is shown; wherein the upper graph is the colony morphology graph cultured at 35 DEG C in blood agar medium for 24 hours under 5% CO2; the lower graph is the colony morphology graph cultured at 35 DEG C in MH agar medium for 24 hours; Figure 2 The typing detection result graph of strain NJGLYY_12739 in example 2 is shown; Figure 3 The typing detection result graph of strain NJGLYY_15701 in example 2 is shown; Figure 4 The whole gene sequencing quality analysis graph of strain NJGLYY_12739 in example 3 is shown; Figure 5 A full-genome sequencing quality analysis graph of strain NJGLYY_15701 in Example 3 is shown; Figure 6 A full-genome sequencing analysis detection result graph of strain NJGLYY_12739 in Example 3 is shown; Figure 7 A full-genome sequencing analysis detection result graph of strain NJGLYY_15701 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 combination 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 primers and the lengths of amplicons are fully considered in view of the technical characteristics of first-generation sequencing. However, the present application re-designs primers and adjusts the lengths of amplicons according to the characteristics of second-generation sequencing platforms.

[0027] Therefore, in one embodiment, the present application provides a primer set for Staphylococcus aureus MLST typing based on a hospital-localized microbial mNGS platform, which comprises a primer pair for amplifying a yqiL gene, a primer pair for amplifying a pta gene, a primer pair for amplifying a tpi gene, a primer pair for amplifying a gmk gene, a primer pair for amplifying a glpF gene, a primer pair for amplifying an aroE gene, and a primer pair for amplifying an arcC gene. The primer pair for amplifying the yqiL gene comprises a forward primer SAU_1.yqiL-F with a nucleotide sequence as shown in SEQ ID NO: 1 and a reverse primer SAU_1.yqiL-R with a nucleotide sequence as shown in SEQ ID NO: 2. yqiL The primer pair for amplifying the pta gene comprises a forward primer SAU_2.pta-F with a nucleotide sequence as shown in SEQ ID NO: 3 and a reverse primer SAU_2.pta-R with a nucleotide sequence as shown in SEQ ID NO: 4. The primer pair for amplifying the tpi gene comprises a forward primer SAU_3.tpi-F with a nucleotide sequence as shown in SEQ ID NO: 5 and a reverse primer SAU_3.tpi-R with a nucleotide sequence as shown in SEQ ID NO: 6. pta The primer pair for amplifying the gmk gene comprises a forward primer SAU_4.gmk-F with a nucleotide sequence as shown in SEQ ID NO: 7 and a reverse primer SAU_4.gmk-R with a nucleotide sequence as shown in SEQ ID NO: 8. The primer pair for amplifying the glpF gene comprises a forward primer SAU_5.glpF-F with a nucleotide sequence as shown in SEQ ID NO: 9 and a reverse primer SAU_5.glpF-R with a nucleotide sequence as shown in SEQ ID NO: 10. tpiThe primer pair of the gene comprises a forward primer SAU_3.tpi-F with a nucleotide sequence as set forth in SEQ ID NO: 5, and a reverse primer SAU_3.tpi-R with a nucleotide sequence as set forth in SEQ ID NO: 6; The amplification gmk The primer pair of the gene comprises a forward primer SAU_4.gmk-F with a nucleotide sequence as set forth in SEQ ID NO: 7, and a reverse primer SAU_4.gmk-R with a nucleotide sequence as set forth in SEQ ID NO: 8; The amplification glpF The primer pair of the gene comprises a forward primer SAU_5.glpF-F with a nucleotide sequence as set forth in SEQ ID NO: 9, and a reverse primer SAU_5.glpF-R with a nucleotide sequence as set forth in SEQ ID NO: 10; The amplification aroE The primer pair of the gene comprises a forward primer SAU_6.aroE-F with a nucleotide sequence as set forth in SEQ ID NO: 11, and a reverse primer SAU_6.aroE-R with a nucleotide sequence as set forth in SEQ ID NO: 12; and The amplification arcC The primer pair of the gene comprises a forward primer SAU_7.arcC-F with a nucleotide sequence as set forth in SEQ ID NO: 13, and a reverse primer SAU_7.arcC-R with a nucleotide sequence as set forth in SEQ ID NO: 14.

[0028] Exemplarily, the specific sequence information of the above primer set is shown in the following table: By constructing multiplex amplification primers, the workload can be greatly reduced. 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 should be optimized to ensure the consistency of the target gene amplification efficiency. Overall, by means of bioinformatics prediction and experimental optimization, a set of efficient and stable multiplex amplification primer combination is constructed to provide qualified DNA library for sequencing, which is another key technical innovation of the present application.

[0029] In another embodiment, a kit for Staphylococcus aureus MLST typing and tracing is provided, which comprises the primer set described above.

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

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

[0032] In yet another embodiment, there is provided use of the primer set or the kit as described above in the library construction of mNGS sequencing for Staphylococcus aureus MLST typing and tracing.

[0033] By applying the primer set and the kit to the library construction of mNGS sequencing for Staphylococcus aureus MLST typing and tracing, it is fully compatible with existing next-generation sequencing platforms, without the need for additional equipment, ensuring efficient operation under existing conditions. In bacterial homology analysis, it will be completed within 24 hours, meeting the timeliness requirements of hospital infection prevention and control. And its accuracy is comparable to traditional MLST or WGS methods.

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

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

[0036] In step 2), the reaction conditions for qPCR amplification reaction on the DNA 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.

[0037] In step 2), the reaction system for qPCR amplification reaction on the DNA to be tested consists of: The primer set, the kit and the method used in the present application are particularly suitable for the instrument for mNGS sequencing, which is PanBios 2000, and the detection server used is CentOS Linux release 7.9.2009 (Core). The combination of the above several can make the time for bacterial typing detection short and the accuracy high.

[0038] The following embodiments are related to and mentioned: 1. mNGS detection platform for Staphylococcus aureus MLST typing and tracing: The instrument used is PanBios 2000, and the server configuration is shown in Table 1.

[0039] Table 1 Instrument model Example 1: Design of primer set According to the traditional MLST system setting, the housekeeping genes of Staphylococcus aureus are: glpF, pta, arcC, tpi, gmk, yqiL, aroE (PubMLST: https: / / pubmlst.org / data). Therefore, according to the technical characteristics of the hospital's next-generation sequencing platform (short sequencing read length, single-end sequencing), the MLST housekeeping gene amplification primers were redesigned, and the length of the amplicon was extended. During the primer design process, attention was paid to optimizing the length, GC content, Tm value and other parameters of the primers to ensure the amplification efficiency and sequencing quality.

[0040] For amplification specificity, first use the Primer-BLAST website to select the "nr" database (the standard non-redundant database) to comprehensively check whether there is inter-species non-specific amplification; after determining the species specificity, further use the Primer-BLAST website, in the "Specificity Check" area, select "S Staphylococcus aureus " as the target species, check for non-specific amplification within the species. Determine that there is no non-specific amplification, that is, it is a suitable primer. Based on the above principles and optimization screening, the primer pair for amplifying the yqiL gene, the primer pair for amplifying the pta gene, the primer pair for amplifying the tpi gene, the primer pair for amplifying the gmk gene, the primer pair for amplifying the glpF gene, the primer pair for amplifying the arcC gene, and the primer pair for amplifying the aroE gene were finally obtained. The sequences of each primer set are shown in SEQ ID NO: 1-14, and the specific nucleotide sequences and amplification fragment information are shown in Table 2: Table 2 PCR amplification primer set Example 2: MLST typing detection of Staphylococcus aureus In this example, two strains of Staphylococcus aureus isolated from the blood 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 blood of patients treated at Nanjing Drum Tower Hospital, and their specific information is shown in Table 3: Table 3 Staphylococcus aureus strain information Figure 1 The culture morphology of two strains NJGLYY_12739 and NJGLYY_15701 is shown; wherein, the upper graph is the colony morphology cultured in blood agar medium at 35℃, 5% CO2 for 24 hours; the lower graph is the colony morphology cultured in MH agar medium at 35℃ for 24 hours.

[0041] 1.2 Extraction of genomic DNA The bacterial genomic DNA extraction kit (DP302, purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.) was used to extract the bacterial genomic DNA according to the operation instruction, and the DNA solution corresponding to the strain was obtained. The bacterial genomic DNA extraction kit (DP302, purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.) was used to extract the bacterial genomic DNA according to the operation instruction, and the DNA solution corresponding to the strain was obtained.

[0042] 1.3 PCR amplification Then, the DNA of the strain to be tested was subjected to PCR amplification reaction using the primer set shown in Table 2 in Example 1, wherein the composition of 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, item number: B639274, purchased from Shengong Bioengineering (Shanghai) Co., Ltd.

[0043] The reaction program is: 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.

[0044] The PCR amplification reaction was carried out according to the above reaction system and program, and the PCR reaction product was obtained.

[0045] 1.4 Product purification The obtained PCR reaction product was purified, which specifically included 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, 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.

[0054] 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.

[0055] 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.

[0056] 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.

[0057] 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.

[0058] 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.

[0059] 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.

[0060] 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.

[0061] 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, Catalog No.: 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.

[0062] 1.7.2 Preparation of DNB system 1 Take 0.2 mL eight-tube or PCR tube, and prepare the reaction system according to the following Table 12 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.

[0063] Table 12 DNB reaction system Table 13 Reaction procedure 1.7.3 Preparation of DNB system 2 Take out the enzyme MB and place it in an ice box, centrifuge briefly to collect the solution to the bottom of the tube, and place it on the ice box for standby. After the PCR reaction, take out the PCR tube, centrifuge briefly to collect the solution to the bottom of the tube, and add the following components shown in Table 14 on ice. Mix well with a vortex shaker, centrifuge briefly to collect the solution to the bottom of the tube, and place it in a PCR instrument for reaction according to the following Table 15.

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

[0065] After the reaction is completed, immediately place it in an ice box, slowly add 20 μL of DNB termination buffer (about 1 drop) with a wide-bore pipette, set the pipette to 100 μL, and slowly suck all the liquid with a wide-bore pipette. Drop by drop, add it to the liquid in the pipette, and repeat 5-8 times. It can be stored at 4°C for standby use (within 24 hours).

[0066] Use the single-stranded DNA (ssDNA) concentration determination kit (fluorescence method) and Qubit 4.0 fluorometer to determine the concentration. Concentration > 8 ng / μL is considered qualified, if not meet the requirements need to re-preparation of DNB.

[0067] Note: a) Because DNB is sticky, it is recommended to take 2 μL for detection. If the number of specimens is large, it is recommended to batch quantification to avoid fluorescence quenching leading to 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 machine 1.8.1 Reagent preparation Take out the slide from the refrigerator, take out the slide from the packaging box but do not open the vacuum packaging bag, and place the slide in a room temperature environment 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 for 5-8 times.

[0069] 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 being opened, 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.

[0070] 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 operation is completed.

[0071] Table 17 Reagent sample amount Finally, the machine operation is performed for sequencing, and the sequencing platform used is Pan Genomics 3000, and the server parameters configured are shown in Table 1.

[0072] 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 Figures 2-3 Table 19 Typing detection results From Tables 19 and Figures 2-3 ​It can be seen that the typing results of the two strains finally detected by the above method are: NJGLYY_12739: 3285 type; NJGLYY_15701: 630 type. The total detection time is 18 hours.

[0073] Example 3: Whole genome sequencing of Staphylococcus aureus The two strains of Staphylococcus aureus in Example 2 were sequenced and typed by whole genome sequencing (completed by Novogene). 1) The whole genome sequencing information of the strains is shown in Table 20 and Figures 4-5 . Table 20 Whole genome sequencing information 2) The final sequencing results are shown in Table 21 and Figures 6-7 .

[0074] Table 21 MLST prediction based on whole genome sequencing data of strains From the above results, the typing detection results of the two strains of Staphylococcus aureus obtained by whole genome sequencing are: NJGLYY_12739 belongs to 3285 type; NJGLYY_15701 belongs to 630 type. 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 based on the technical essence of the present application are still within the scope of the technical solution of the present application.

Claims

1. A primer set for MLST typing of Staphylococcus aureus based on a hospital-based microbial mNGS platform, characterized in that: Including amplification yqQ Primer pairs for gene amplification pta Primer pairs for gene amplification tpi Primer pairs for gene amplification gmk Primer pairs for gene amplification glpF Primer pairs for gene amplification aroE Primer pairs and amplification of genes arcC primer pairs for the genes; The amplification yqQ The primer pair for the gene includes a forward primer SAU_1.yqiL-F having a nucleotide sequence as shown in SEQ ID NO: 1, and a reverse primer SAU_1.yqiL-R having a nucleotide sequence as shown in SEQ ID NO: 2; The amplification pta The primer pair for the gene includes a forward primer SAU_2.pta-F having a nucleotide sequence as shown in SEQ ID NO: 3, and a reverse primer SAU_2.pta-R having a nucleotide sequence as shown in SEQ ID NO: 4; The amplification tpi The primer pair for the gene includes a forward primer SAU_3.tpi-F having a nucleotide sequence as shown in SEQ ID NO: 5, and a reverse primer SAU_3.tpi-R having a nucleotide sequence as shown in SEQ ID NO: 6; The amplification gmk The primer pair for the gene includes a forward primer SAU_4.gmk-F having a nucleotide sequence as shown in SEQ ID NO: 7, and a reverse primer SAU_4.gmk-R having a nucleotide sequence as shown in SEQ ID NO: 8; The amplification glpF The primer pair for the gene includes a forward primer SAU_5.glpF-F having a nucleotide sequence as shown in SEQ ID NO: 9, and a reverse primer SAU_5.glpF-R having a nucleotide sequence as shown in SEQ ID NO: 10; The amplification aroE The primer pair for the gene includes a forward primer SAU_6.aroE-F having a nucleotide sequence as shown in SEQ ID NO: 11 and a reverse primer SAU_6.aroE-R having a nucleotide sequence as shown in SEQ ID NO: 12; and The amplification arcC The primer pair for the gene includes a forward primer SAU_7.arcC-F whose nucleotide sequence is shown in SEQ ID NO: 13, and a reverse primer SAU_7.arcC-R whose nucleotide sequence is shown in SEQ ID NO:

14.

2. A kit for MLST typing of Staphylococcus aureus 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 of claim 1 or the kit of claim 2 or 3 in constructing a Staphylococcus aureus mNGS sequencing library for MLST typing of Staphylococcus aureus.

5. A method for constructing an mNGS sequencing library for MLST typing of Staphylococcus aureus, 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 Staphylococcus aureus.

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