Primer group and method for klebsiella pneumoniae 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 Klebsiella pneumoniae MLST typing primer group and method based on a hospital localized microbiological mNGS platform. The primer group comprises primer pairs for amplifying an rpoB gene, an infB gene, a pgi gene, a phoE gene, a gapA gene, an mdh gene and a tonB 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 primer group for Klebsiella pneumoniae MLST housekeeping gene amplification is redesigned, and amplicons are adaptive to the sequencing working principle of the existing next-generation sequencing platform in the hospital by optimizing the length and position of the amplicons. By utilizing the primer group, the kit or the method, the genetic relationship between hospital infection pathogenic bacteria can be quickly and accurately judged (within 24 hours), the timeliness and scientificity of infection prevention and control are remarkably improved, and the primer group, the kit or the method have a wide clinical application prospect.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of microorganism detection, and particularly relates to a primer set and method for Klebsiella pneumoniae 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" according to whether the bacteria are of the same type to determine 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), etc. 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 conserved gene sites, and these sequences are internationally agreed and known. MLST relies on the difference in bacterial conserved 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 conserved 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 transmission 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 a 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 the amplification product is sent to a sequencing company for detection, the data obtained is uploaded to an overseas website for query, and finally the ST type is determined. The process has the following disadvantages: (1) equipment and technology limitation: there is generally no related equipment and technology in the hospital and the CDC, and only a third-party institution can be sent for detection. The logistics time needs to be consumed in the process of sending, which seriously affects the timeliness. (2) result transmission and manual operation: after the sequencing is completed, the detection result is transmitted through the website or the email, but the final splicing and comparison still need manual operation, which is time-consuming and laborious and is 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, but the second-generation sequencing for scientific research also has the problem of sample outsourcing, and the sequencing period is long, which is 3 days, and cannot meet the actual needs of clinical infection prevention and control. (4) data security: the determination and query of the ST type need to upload the data to an overseas website, and there is a risk of domestic data leakage. From another aspect, if from the perspective of scientific research, there is also a method of performing cgMLST analysis through whole genome sequencing. The method produces a large amount of data, and the result reaches 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 the bacterial MLST typing is needed in the 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 diseases within 24 hours, some domestic first-class hospitals actively construct a localized second-generation sequencing platform, so as to carry out pathogenic microorganism second-generation sequencing detection service in the hospital (instead of outsourcing to a sequencing company). Although the purpose of constructing the localized sequencing platform is to carry out pathogenic microorganism detection work, it also provides material basis and technical support for us to utilize the platform, carry out homology analysis of nosocomial infection bacteria through technical innovation. SUMMARY

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

[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 Klebsiella pneumoniae MLST typing based on a hospital localized microorganism mNGS platform, which comprises a primer pair for amplifying rpoB the gene, and a primer pair for amplifyinginfB a pair of primers for amplifying a gene pgi a pair of primers for amplifying a gene phoE a pair of primers for amplifying a gene gapA a pair of primers for amplifying a gene mdh a pair of primers for amplifying a gene tonB a pair of primers for amplifying a gene The pair of primers for amplifying the rpoB gene comprises a forward primer KP1_rpoB_F with a nucleotide sequence as shown in SEQ ID NO: 1, and a reverse primer KP1_rpoB_R with a nucleotide sequence as shown in SEQ ID NO: 2. The pair of primers for amplifying the infB gene comprises a forward primer KP2_infB_F with a nucleotide sequence as shown in SEQ ID NO: 3, and a reverse primer KP2_infB_R with a nucleotide sequence as shown in SEQ ID NO: 4. The pair of primers for amplifying the pgi gene comprises a forward primer KP3_pgi_F with a nucleotide sequence as shown in SEQ ID NO: 5, and a reverse primer KP3_pgi_R with a nucleotide sequence as shown in SEQ ID NO: 6. The pair of primers for amplifying the phoE gene comprises a forward primer KP4_phoE_F with a nucleotide sequence as shown in SEQ ID NO: 7, and a reverse primer KP4_phoE_R with a nucleotide sequence as shown in SEQ ID NO: 8. The pair of primers for amplifying the gapA gene comprises a forward primer KP5_gapA_F with a nucleotide sequence as shown in SEQ ID NO: 9, and a reverse primer KP5_gapA_R with a nucleotide sequence as shown in SEQ ID NO: 10. The pair of primers for amplifying the mdh gene comprises a forward primer KP6_mdh_F with a nucleotide sequence as shown in SEQ ID NO: 11, and a reverse primer KP6_mdh_R with a nucleotide sequence as shown in SEQ ID NO: 12. The pair of primers for amplifying the tonB gene comprises a forward primer KP7_tonB_F with a nucleotide sequence as shown in SEQ ID NO: 13, and a reverse primer KP7_tonB_R with a nucleotide sequence as shown in SEQ ID NO: 14.

[0011] The second aspect of the present application provides a kit for Klebsiella pneumoniae MLST typing based on a hospital-localized microbial mNGS platform, which comprises 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 a method for mNGS sequencing library construction for K. pneumoniae MLST typing source tracing.

[0015] The fourth aspect of the present application provides a method for mNGS sequencing library construction for K. pneumoniae MLST typing source 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.

[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 K. pneumoniae MLST typing source tracing.

[0020] By means of the above technical solution, the present application has at least the following advantages: The present application re-designs the amplification primer for Klebsiella pneumoniae MLST housekeeping genes according to the technical characteristics of 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 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 a specific second-generation sequencing platform is used for sequencing, which can realize rapid and accurate homology analysis of Klebsiella pneumoniae, 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 infection pathogens, 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 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 primer design and sequencing process, so that bacterial MLST typing detection can be performed using the remaining positions of the chip while daily clinical routine mNGS detection is carried out. 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 the contents of the specification can be implemented as follows. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 The figure shows the typing detection result of strain NJGLYY3598 in Example 2; Figure 2 The figure shows the typing detection result of strain NJGLYY4165 in Example 2; Figure 3 The figure shows the whole gene sequencing quality analysis of strain NJGLYY3598 in Example 3; Figure 4 The figure shows the whole gene sequencing quality analysis of strain NJGLYY4165 in Example 3; Figure 5 The figure shows the whole gene sequencing circle of strain NJGLYY3598 in Example 3; Figure 6A circle diagram showing whole genome sequencing of strain NJGLYY4165 in Example 3 is shown; Figure 7 A diagram showing the detection results of whole genome sequencing analysis of strain NJGLYY3598 in Example 3 is shown; Figure 8 A diagram showing the detection results of whole genome sequencing analysis of strain NJGLYY4165 in Example 3 is shown. DETAILED DESCRIPTION

[0025] In order to make the technical means, creative features, purposes and effects achieved 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 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 Klebsiella pneumoniae MLST typing based on a hospital-localized microbial mNGS platform, comprising primer pairs for amplifying rpoB genes, primer pairs for amplifying infB genes, primer pairs for amplifying pgi genes, primer pairs for amplifying phoE genes, primer pairs for amplifying gapA genes, primer pairs for amplifying mdh genes, and primer pairs for amplifying tonB genes. The primer pairs for amplifying rpoB genes comprise a forward primer KP1_rpoB_F with a nucleotide sequence as shown in SEQ ID NO: 1, and a reverse primer KP1_rpoB_R with a nucleotide sequence as shown in SEQ ID NO: 2. The primer pairs for amplifying infB genes comprise a forward primer KP2_infB_F with a nucleotide sequence as shown in SEQ ID NO: 3, and a reverse primer KP2_infB_R with a nucleotide sequence as shown in SEQ ID NO: 4. The primer pairs for amplifying pgiThe primer pair of the gene comprises a forward primer KP3_pgi_F with a nucleotide sequence as shown in SEQ ID NO: 5, and a reverse primer KP3_pgi_R with a nucleotide sequence as shown in SEQ ID NO: 6; The amplification phoE The primer pair of the gene comprises a forward primer KP4_phoE_F with a nucleotide sequence as shown in SEQ ID NO: 7, and a reverse primer KP4_phoE_R with a nucleotide sequence as shown in SEQ ID NO: 8; The amplification gapA The primer pair of the gene comprises a forward primer KP5_gapA_F with a nucleotide sequence as shown in SEQ ID NO: 9, and a reverse primer KP5_gapA_R with a nucleotide sequence as shown in SEQ ID NO: 10; The amplification mdh The primer pair of the gene comprises a forward primer KP6_mdh_F with a nucleotide sequence as shown in SEQ ID NO: 11, and a reverse primer KP6_mdh_R with a nucleotide sequence as shown in SEQ ID NO: 12; and The amplification tonB The primer pair of the gene comprises a forward primer KP7_tonB_F with a nucleotide sequence as shown in SEQ ID NO: 13, and a reverse primer KP7_tonB_R with a nucleotide sequence as shown in SEQ ID NO: 14.

[0028] Exemplarily, the specific sequence information of the above primer group is shown in the following table: The original MLST primer and the length of the amplification product are 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, through the two aspects 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 Klebsiella pneumoniae MLST typing and tracing is provided, which comprises the primer group described above.

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

[0031] In a further optimization 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 previously described in the mNGS sequencing library construction of Klebsiella pneumoniae for Klebsiella pneumoniae MLST typing.

[0033] By applying the primer set and the kit to the mNGS sequencing library construction of Klebsiella pneumoniae for Klebsiella pneumoniae MLST typing, it is fully adapted to the existing next-generation sequencing platform, without the need for additional purchase of equipment, ensuring efficient operation under existing conditions. On 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 mNGS sequencing library construction of Klebsiella pneumoniae for Klebsiella pneumoniae 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 previously described; 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 used in mNGS sequencing, which is Panbion 2000, and the detection server used is CentOS Linux release 7.9.2009 (Core). The combination of the above several can make the time used for bacterial typing detection short and the accuracy high.

[0038] The following examples are related to and mentioned: 1. mNGS detection platform for Klebsiella pneumoniae MLST typing: The instrument used is: Pan Biosystems 2000, and the server configuration is shown in Table 1.

[0039] Table 1 Instrument model Example 1: Design of primer set According to the conventional MLST system, the housekeeping genes of Klebsiella pneumoniae are: gapA, infB, mdh, pgi, phoE, rpoB, tonB (PubMLST: https: / / pubmlst.org / data). Therefore, according to the technical characteristics of the hospital's second-generation sequencing platform (short sequencing read length, single-end sequencing), the MLST housekeeping gene amplification primers are redesigned, and the length of the amplicon is extended. During the primer design process, attention is 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 standard non-redundant 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 the target species as "Klebsiella pneumoniae", and check for non-specific amplification within the species. Determine that there is no non-specific amplification, which is a suitable primer. Based on the above principles and optimization screening, the primer pair for amplifying the Klebsiella pneumoniae gene, the primer pair for amplifying the rpoB gene, the primer pair for amplifying the infB gene, the primer pair for amplifying the pgi gene, the primer pair for amplifying the phoE gene, the primer pair for amplifying the gapA gene, the primer pair for amplifying the mdh gene, and the amplification tonB The primer pairs for amplifying the gene, and the amplification Table 2 PCR amplification primer set Example 2: MLST typing detection of Klebsiella pneumoniae In this example, two strains of Klebsiella pneumoniae isolated from the wound secretions and 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 the present example were isolated from wound exudates and sputum of patients treated in Nanjing Gulou Hospital, and the specific information is shown in Table 3: Table 3 Strain information of Klebsiella pneumoniae 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.

[0041] 1.3 PCR amplification Subsequently, 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 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, item number: B639274, purchased from Shengong Bioengineering (Shanghai) Co., Ltd.

[0042] The reaction program was 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.

[0043] The PCR amplification reaction was carried out according to the above reaction system and program, and the PCR reaction product was obtained.

[0044] 1.4 Product purification The obtained PCR reaction product was purified, which specifically included the following steps: Add 45 μL of purified DNA beads to 50 μL of PCR product. Vortex or pipette 10 times to mix thoroughly. Incubate at room temperature for 5 min. Briefly centrifuge to collect the mixture at the bottom of the tube. Place the PCR tube on a magnetic rack and let it stand for about 3-5 min until the liquid in the tube becomes clear. Carefully aspirate all the liquid. Add 200 μL of 80% ethanol (freshly prepared), let it stand for 30 s, and carefully aspirate all the liquid. Note that when aspirating the 80% ethanol, the action should be slow and thorough to avoid leaving any droplets on the tube wall. If a small amount of liquid remains on the tube wall, briefly centrifuge the tube, place it back on the magnetic rack, and aspirate the liquid at the bottom of the tube using a small-capacity pipette. Open the cap and let it dry for 3-5 min until the magnetic beads are no longer reflective and show slight cracks (i.e., slight cracks are observed in the bead cluster). Add 32.5 μL of Buffer EB, close the cap, remove the PCR tube from the magnetic rack, vortex to mix, incubate at room temperature for 2 min, and briefly centrifuge to collect the solution at the bottom of the tube. Place the PCR tube on the magnetic rack and incubate for about 2 min until fully magnetically attracted and the liquid is clear. Aspirate 30 μL of the liquid into a new 1.5 mL centrifuge tube, being careful not to aspirate the magnetic beads, to obtain the purified product.

[0045] 1.5 Library Construction 1.5.1 Fragmentation and End Repair Remove the host-free Q-Buffer reagent, thaw it briefly, add 120 μL of Buffer EB, vortex to mix, and prepare the Q-Buffer dilution. Place it on ice for later use. Next, prepare the fragmentation and end-repair system as shown in Table 5. After preparation, pipette and mix 10 times, briefly centrifuge to collect the mixture at the bottom of the tube, and place it on ice for later use. Finally, set the PCR reaction program as shown in Table 6. After setting, incubate the prepared fragmentation and end-repair system in a PCR instrument. After incubation, obtain the fragmentation and end-repair products and place them on ice for later use.

[0046] Table 5 Fragmentation and End-of-Life Repair System Note: The amount of DNA used in the table is ≤5 ng, and the volume is ≤30 μL. The reagent preparation for this step requires double verification by two people. The reagent preparation can proceed only after the verification is complete.

[0047] Table 6 Reaction Procedure Note: This step requires two people to verify the PCR procedure. After confirming that there are no errors, the PCR reaction can be performed.

[0048] 1.5.2 Ligation (using a universal kit prepared by a sequencing reaction, REF (catalog number): 2102, purchased from Tianjin Jinkey Medical Technology Co., Ltd.) First, prepare the ligation system as shown in Table 7, after preparation, mix 10 times by pipette blowing, centrifuge briefly to collect the mixture to the bottom of the tube, and place on ice for standby. Then set the PCR reaction program as shown in Table 8, after setting, place the prepared ligation system in the PCR instrument for incubation, after incubation, obtain the ligation product, and place on ice for standby.

[0049] Table 7 Ligation system Note: The UDB Adapter stock solution in the table is 10 μM, which needs to be diluted before use; During the experiment, the reagents need to be double-checked by two persons, and the reagent preparation can be carried out after confirming that there is no mistake.

[0050] Table 8 Reaction program Note: This step needs to be double-checked by two persons, and the PCR reaction can be carried out after confirming that there is no mistake.

[0051] 1.5.3 Purification of ligation product Add 60 μL of DNA purification magnetic beads (DNA purification magnetic beads are stored in a 4 ℃ refrigerator, and need to be taken out for 30 min to balance to room temperature before use, and vortexed before use) to the above obtained 100 μL ligation product, mix thoroughly by pipette blowing for 10 times, incubate at room temperature for 5 min, and centrifuge briefly to collect the mixture to the bottom of the tube. Then place the PCR tube on the magnetic stand, stand for about 3-5 min, and carefully discard all the liquid when the liquid in the tube is clear. Add 200 μL of 80% ethanol (freshly prepared), stand for 30 s, and carefully discard all the liquid; It should be noted that the 80% ethanol should be discarded slowly, and the liquid droplets should be discarded 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, centrifuge the centrifugal tube briefly, place it on the magnetic stand again, and mix the liquid in the bottom of the tube with a small volume pipette. Open the cap and dry for 3-5 min until the magnetic beads have no reflection and slight cracks (i.e. observe the slight cracks of the magnetic bead group). Then add 23 μL of Buffer EB, remove the PCR tube from the magnetic stand, vortex to mix, stand at room temperature for 2 min, and centrifuge briefly to collect the solution to the bottom of the tube. Finally, place the PCR tube on the magnetic stand, stand for 2 min until the liquid is fully magnetized and clear, and aspirate 21 μL of liquid into a new PCR tube, without aspirating the magnetic beads, to obtain the purified 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 was 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 were calculated according to the library concentration and the required data volume. The libraries were arranged in the mixing order, and the corresponding volume of each library was drawn into a 1.5 mL EP tube according to the calculated mixing volume, mixed well, and the solution was 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 were used to determine the library concentration, and the results were filled into the "pooling measured concentration" in the mixing operation table. The measured mixing concentration was compared with the theoretical mixing concentration, and the difference between the two was 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 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.) were taken out and placed in an ice box for about 0.5 h, then mixed well using a vortex shaker for 5 s, and centrifuged briefly and placed on ice for standby.

[0061] 1.7.2 Preparation of DNB system 1 A 0.2 mL eight-tube or PCR tube was taken, and the reaction system was prepared on ice according to the system shown in Table 12 below. Mix well 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 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.

[0063] 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.

[0064] 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).

[0065] 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.

[0066] 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.

[0067] 1.8 Sequencing on machine 1.8.1 Reagent preparation Take the slide out of the refrigerator, take the slide out of the packaging box but do not open the vacuum packaging bag, and place the slide 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.

[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 Table 19 and Figures 1-2 Table 19 Typing detection results Table 19 and Figures 1-2 ​It can be seen that the typing results of the two strains of bacteria finally detected by the above method are: NJGLYY3598: 1779 type; NJGLYY4165: 11 type. The total detection time is: 18 hours.

[0072] Example 3: Whole genome sequencing of Klebsiella pneumoniae The two strains of Klebsiella pneumoniae in Example 2 were sequenced and typed by whole genome sequencing (completed by Novogene Co., Ltd.), wherein: 1) The whole genome sequencing information of the strains is shown in Table 20 and Figures 3-4 . Table 20 Whole genome sequencing information 2) The whole genome sequencing circle diagram of the two strains is shown in Figures 5-6 .

[0073] 3) The final sequencing results are shown in Table 21 and Figures 7-8 .

[0074] Table 21 MLST prediction based on whole genome sequencing data of strains From the above results, it can be seen that the typing detection results of the two strains of Klebsiella pneumoniae obtained by whole genome sequencing are: NJGLYY3598 belongs to ST1779 type; NJGLYY4165 belongs to ST11 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, 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 according to 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 Klebsiella pneumoniae MLST typing based on a hospital-localized microbial mNGS platform, characterized in that, comprising a primer pair, amplification rpoB a primer pair, amplification infB a primer pair, amplification pgi a primer pair, amplification phoE a primer pair, amplification gapA a primer pair, amplification mdh a primer pair, amplification tonB a primer pair The amplification rpoB The primer pair of the gene comprises a forward primer KP1_rpoB_F with a nucleotide sequence as shown in SEQ ID NO: 1, and a reverse primer KP1_rpoB_R with a nucleotide sequence as shown in SEQ ID NO: 2; The amplification infB The primer pair of the gene comprises a forward primer KP2_infB_F with a nucleotide sequence as shown in SEQ ID NO: 3, and a reverse primer KP2_infB_R with a nucleotide sequence as shown in SEQ ID NO: 4; The amplification pgi The primer pair of the gene comprises a forward primer KP3_pgi_F with a nucleotide sequence as set forth in SEQ ID NO: 5, and a reverse primer KP3_pgi_R with a nucleotide sequence as set forth in SEQ ID NO: 6; The amplification phoE The primer pair of the gene comprises a forward primer KP4_phoE_F with a nucleotide sequence as set forth in SEQ ID NO: 7, and a reverse primer KP4_phoE_R with a nucleotide sequence as set forth in SEQ ID NO: 8; The amplification gapA The primer pair of the gene comprises a forward primer KP5_gapA_F with a nucleotide sequence as set forth in SEQ ID NO: 9, and a reverse primer KP5_gapA_R with a nucleotide sequence as set forth in SEQ ID NO: 10; The amplification mdh The primer pair for the gene includes a forward primer KP6_mdh_F having a nucleotide sequence as set forth in SEQ ID NO: 11, and a reverse primer KP6_mdh_R having a nucleotide sequence as set forth in SEQ ID NO: 12; and The amplification tonB The primer pair of the gene comprises a forward primer KP7_tonB_F having a nucleotide sequence as set forth in SEQ ID NO: 13, and a reverse primer KP7_tonB_R having a nucleotide sequence as set forth in SEQ ID NO:

14.

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

3. The kit of claim 2, wherein The kit further comprises 2X TaqMan FastqPCR Master Mix.

4. Use of the primer set of claim 1 or the kit of claim 2 or 3 in the library construction of mNGS sequencing of Klebsiella pneumoniae for K. pneumoniae MLST typing.

5. A method for sequencing library construction of mNGS for Klebsiella pneumoniae MLST typing, characterized in that, comprising the following steps: 1) extracting DNA of the sample to be tested; 2) performing qPCR amplification reaction on the DNA of the sample to be tested extracted in step 1) using the kit of claim 2 or 3; 3) adding mNGS sequencing adapters to both ends of the amplification reaction product of step 2) to obtain an mNGS sequencing library.

6. The method of claim 5, wherein, In step 1), bacterial genomic DNA extraction kit is used to extract DNA from the sample to be tested.

7. The method of claim 5, wherein, In step 2), the reaction conditions for performing qPCR amplification reaction on the DNA of 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.

8. The method of claim 5, wherein, In step 2), the reaction system for performing qPCR amplification reaction on the DNA of the sample to be tested comprises: 。 9. The method of claim 5, wherein, The instrument for mNGS sequencing is PanGenome 2000, and the detection server used is CentOS Linux release 7.9.2009 (Core).

10. Use of the primer set of claim 1 or the kit of claim 2 or 3 or the method of any one of claims 5-9 in the preparation of a reagent for detecting K. pneumoniae MLST typing.

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