Primer group and method for acinetobacter baumannii MLST typing based on hospital localized microorganism mNGS platform

Abstract

The invention belongs to the technical field of microbiological detection, and particularly relates to an acinetobacter baumannii MLST typing primer group and method based on a hospital localized microbiological mNGS platform. The primer group comprises a primer pair for amplifying a gyrB gene, a gpi gene, a gdhB gene, a recA gene, a cpn60 gene, a gltA gene and an rpoD gene, and the 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, an amplification primer group for the acinetobacter baumannii MLST housekeeping gene is redesigned, and amplicons are adaptive to the sequencing principle of the existing next-generation sequencing platform in the hospital by optimizing the length and the 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 Acinetobacter baumannii 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. Hospitals 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 transmission. 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 MLST typing of Acinetobacter baumannii based on a hospital localized microorganism mNGS platform, so as to realize rapid and accurate typing detection of Acinetobacter baumannii.

[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 MLST typing of Acinetobacter baumannii based on a hospital localized microorganism mNGS platform, which comprises a primer pair for amplifying gyrB the gene, and a primer pair for amplifyinggpi a pair of primers for amplifying a gene gdhB a pair of primers for amplifying a gene recA a pair of primers for amplifying a gene cpn60 a pair of primers for amplifying a gene gltA a pair of primers for amplifying a gene rpoD a pair of primers for amplifying a gene the amplification gyrB a pair of primers for amplifying a gene includes a forward primer Abaumannii_1.gyrB-F having a nucleotide sequence as shown in SEQ ID NO: 1, and a reverse primer Abaumannii_1.gyrB-R having a nucleotide sequence as shown in SEQ ID NO: 2; the amplification gpi a pair of primers for amplifying a gene includes a forward primer Abaumannii_2.gpi-F having a nucleotide sequence as shown in SEQ ID NO: 3, and a reverse primer Abaumannii_2.gpi-R having a nucleotide sequence as shown in SEQ ID NO: 4; the amplification gdhB a pair of primers for amplifying a gene includes a forward primer Abaumannii_3.gdhB-F having a nucleotide sequence as shown in SEQ ID NO: 5, and a reverse primer Abaumannii_3.gdhB-R having a nucleotide sequence as shown in SEQ ID NO: 6; the amplification recA a pair of primers for amplifying a gene includes a forward primer Abaumannii_4.recA-F having a nucleotide sequence as shown in SEQ ID NO: 7, and a reverse primer Abaumannii_4.recA-R having a nucleotide sequence as shown in SEQ ID NO: 8; the amplification cpn60 a pair of primers for amplifying a gene includes a forward primer Abaumannii_5.cpn60-F having a nucleotide sequence as shown in SEQ ID NO: 9, and a reverse primer Abaumannii_5.cpn60-R having a nucleotide sequence as shown in SEQ ID NO: 10; the amplification gltA a pair of primers for amplifying a gene includes a forward primer Abaumannii_6.gltA-F having a nucleotide sequence as shown in SEQ ID NO: 11, and a reverse primer Abaumannii_6.gltA-R having a nucleotide sequence as shown in SEQ ID NO: 12; and the amplification rpoDThe primer pair of the gene comprises a forward primer Abaumannii_7.rpoD-F having a nucleotide sequence as shown in SEQ ID NO: 13, and a reverse primer Abaumannii_7.rpoD-R having a nucleotide sequence as shown in SEQ ID NO: 14.

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

[0012] In a preferred embodiment of the present 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 Acinetobacter baumannii MLST typing traceability of Acinetobacter baumannii mNGS sequencing library construction.

[0015] The fourth aspect of the present application provides an mNGS sequencing library construction method for Acinetobacter baumannii MLST typing traceability, comprising the following steps: 1) extracting DNA from a 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 composition for performing qPCR amplification reaction on the DNA to be tested is as follows: In a preferred embodiment of the present application, the instrument used for mNGS sequencing is Nanopore 2000, and the detection server used is CentOS Linux release 7.9.2009 (Core).

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

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

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

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

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

[0024] Figure 1Colony morphology of two strains of Acinetobacter baumannii NJGLYY14991 and NJGLYY15376 is shown; wherein, the upper graph is the colony morphology chart cultured for 24 hours at 35℃ in blood agar medium with 5% CO2; the lower graph is the colony morphology chart cultured for 24 hours at 35℃ in MH agar medium; Figure 2 The figure of the typing detection result of the strain NJGLYY14991 in Example 2 is shown; Figure 3 The figure of the typing detection result of the strain NJGLYY15376 in Example 2 is shown; Figure 4 The figure of the whole gene sequencing quality analysis of the strain NJGLYY14991 in Example 3 is shown; Figure 5 The figure of the whole gene sequencing quality analysis of the strain NJGLYY15376 in Example 3 is shown; Figure 6 The figure of the whole gene sequencing analysis detection result of the strain NJGLYY14991 in Example 3 is shown; Figure 7 The figure of the whole gene sequencing analysis detection result of the strain NJGLYY15376 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 a 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 skilled in the art without creative labor fall within the protection scope of the present application.

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

[0027] Therefore, in one embodiment, the present application provides a primer set for Acinetobacter baumannii MLST typing based on a hospital localized microbial mNGS platform, which comprises a primer pair for amplifying gyrB a gene, a primer pair for amplifying gpi a gene, a primer pair for amplifying gdhB a gene, a primer pair for amplifying recA a gene, a primer pair for amplifying cpn60 a gene, and a primer pair for amplifying rpoDa primer pair of a gene; the amplification gyrB The primer pair of the gene includes a forward primer Abaumannii_1.gyrB-F having a nucleotide sequence as shown in SEQ ID NO: 1, and a reverse primer Abaumannii_1.gyrB-R having a nucleotide sequence as shown in SEQ ID NO: 2; the amplification gpi The primer pair of the gene includes a forward primer Abaumannii_2.gpi-F having a nucleotide sequence as shown in SEQ ID NO: 3, and a reverse primer Abaumannii_2.gpi-R having a nucleotide sequence as shown in SEQ ID NO: 4; the amplification gdhB The primer pair of the gene includes a forward primer Abaumannii_3.gdhB-F having a nucleotide sequence as shown in SEQ ID NO: 5, and a reverse primer Abaumannii_3.gdhB-R having a nucleotide sequence as shown in SEQ ID NO: 6; the amplification recA The primer pair of the gene includes a forward primer Abaumannii_4.recA-F having a nucleotide sequence as shown in SEQ ID NO: 7, and a reverse primer Abaumannii_4.recA-R having a nucleotide sequence as shown in SEQ ID NO: 8; the amplification cpn60 The primer pair of the gene includes a forward primer Abaumannii_5.cpn60-F having a nucleotide sequence as shown in SEQ ID NO: 9, and a reverse primer Abaumannii_5.cpn60-R having a nucleotide sequence as shown in SEQ ID NO: 10; the amplification gltA The primer pair of the gene includes a forward primer Abaumannii_6.gltA-F having a nucleotide sequence as shown in SEQ ID NO: 11, and a reverse primer Abaumannii_6.gltA-R having a nucleotide sequence as shown in SEQ ID NO: 12; and the amplification rpoD The primer pair of the gene includes a forward primer Abaumannii_7.rpoD-F having a nucleotide sequence as shown in SEQ ID NO: 13, and a reverse primer Abaumannii_7.rpoD-R having a nucleotide sequence as shown in SEQ ID NO: 14.

[0028] Exemplarily, the specific sequence information of the above primer set is shown in the following table: The workload can be greatly reduced by constructing multiplex amplification primers. When designing multiplex amplification primers, cross-reaction or non-specific amplification between primers should be avoided as much as possible. In addition, the amplification conditions such as primer concentration and annealing temperature need to be optimized to ensure the consistency of the target gene amplification efficiency. In general, through the two aspects of bioinformatics prediction and experimental optimization, constructing a set of efficient and stable multiplex amplification primer combination provides qualified DNA library for sequencing, which is another key technical innovation of the application.

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

[0030] As a preference, 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, the application provides the use of the primer set or kit as described above in the mNGS sequencing library construction of Acinetobacter baumannii for Acinetobacter baumannii MLST typing and tracing.

[0033] By applying the primer set and kit to the mNGS sequencing library construction of Acinetobacter baumannii for Acinetobacter baumannii 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, a method for mNGS sequencing library construction of Acinetobacter baumannii for Acinetobacter baumannii MLST typing and tracing is provided, comprising the following steps: 1) Extracting DNA from the sample to be tested; 2) Using the kit as described above to perform qPCR amplification reaction on the DNA extracted from the sample to be tested in step 1); 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 of the test sample DNA are as follows: 94°C for 2 min, cycle number 1; 94°C for 30 s, cycle number 35; 50°C for 1 min; 72°C for 30 s; 72°C for 5 min, cycle number 1; 4°C forever.

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

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

[0039] Table 1 Instrument Model Example 1: Design of primer sets The present invention is based on the traditional MLST system, and the housekeeping genes of Acinetobacter baumannii are: cpn60, gpi, gltA, gdhB, recA, gyrB, rpoD (PubMLST: https: / / pubmlst.org / data). Therefore, based on the technical characteristics of the in-house next-generation sequencing platform (short sequencing read length, single-end sequencing), MLST housekeeping gene amplification primers were redesigned to extend the amplicon length. During the primer design process, attention was paid to optimizing parameters such as primer length, GC content, and Tm value to ensure amplification efficiency and sequencing quality.

[0040] For amplification specificity, first use the Primer-BLAST website and select the "nr" database (the standard non-redundant database) to comprehensively check whether there is non-specific amplification between species; after confirming the species specificity, further use the Primer-BLAST website and select " Acinetobacter baumannoii gyrB "As the target species, check the non-specific amplification in the species. If there is no non-specific amplification, it is the appropriate primer. Based on the above principles and optimized screening, the amplification was finally obtained.gpi Primer pairs, amplification of genes gdhB Primer pairs, amplification of genes recA Primer pairs, amplification of genes cpn60 Primer pairs, amplification of genes gltA Primer pairs, amplification of genes rpoD Primer pairs and amplification of genes Figure 1 Primer pairs of genes, each primer group sequence is shown as SEQ ID NO: 1-14, and the specific nucleotide sequence and amplification fragment information are shown in Table 2: Table 2 PCR amplification primer group Example 2: MLST typing detection of Acinetobacter baumannii In this example, two strains of Acinetobacter baumannii isolated from the blood of a subject were used as test samples, and the primer group determined in Example 1 was used for MLST typing detection of 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 the specific information is shown in Table 3: Table 3 Acinetobacter baumannii strain information As shown in Figures 2-3 , are the colony morphologies of two strains of Acinetobacter baumannii NJGLYY14991 and NJGLYY15376; wherein, the upper graph is the colony morphology after 24 hours of culture at 35°C in blood agar medium with 5% CO2; and the lower graph is the colony morphology after 24 hours of culture at 35°C in MH agar medium.

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

[0042] 1.3 PCR amplification Subsequently, the primer group shown in Table 2 in Example 1 was used for PCR amplification reaction of the DNA of the test strain, 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.

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

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

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

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

[0047] Table 5 Fragmentation and end-repair system Note: The amount of DNA input in the table is ≤5 ng, and the input volume is ≤30 μL; the reagent configuration for this step requires double checking by two persons, and the reagent configuration can be performed after confirming that there is no error.

[0048] Table 6 Reaction procedure Note: This step requires double checking of the PCR program by two persons, and the PCR reaction can be performed after confirming that there is no error.

[0049] 1.5.2 Adapter ligation (using sequencing reaction to prepare universal kit, REF (item number): 2102, purchased from Tianjin Jinkey Medical Technology Co., Ltd.) First, prepare the adapter ligation system as shown in Table 7, mix well by pipette blowing for 10 times after configuration, 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 adapter ligation system in the PCR instrument for incubation, after incubation, obtain the adapter ligation product, and place on ice for standby.

[0050] Table 7 Adapter ligation system Note: The UDB Adapter stock solution in the table is 10 μM, which needs to be diluted before use; double checking of the reagent by two persons is required during the experiment, and the reagent configuration can be performed after confirming that there is no error.

[0051] Table 8 Reaction procedure Note: This step requires double checking of the PCR program by two persons, and the PCR reaction can be performed after confirming that there is no error.

[0052] 1.5.3 Purification of adapter ligation product To the 100 μL of the ligation product, 60 μL of DNA purification magnetic beads (DNA purification magnetic beads were stored in a 4 ℃ refrigerator and needed to be taken out for 30 min to balance to room temperature, and were vortexed and mixed before use) were added, and the mixture was mixed by blowing 10 times with a pipette, incubated at room temperature for 5 min, and then centrifuged briefly to collect the mixture at the bottom of the tube. Then the PCR tube was placed on a magnetic stand and stood for about 3-5 min, and when the liquid in the tube was clear, the liquid was carefully removed. 200 μL of 80% ethanol (freshly prepared) was added, and the mixture was stood for 30 s, and then the liquid was carefully removed. It should be noted that the 80% ethanol should be removed slowly, and the liquid droplets should be removed 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 centrifugal tube can be briefly centrifuged, placed on the magnetic stand again, and the liquid at the bottom of the tube can be removed with a small volume pipette. The cap was opened and dried for 3-5 min, until the magnetic beads were no longer shiny and had slight cracks (i.e., the magnetic bead group had slight cracks). Then 23 μL of Buffer EB was added, the PCR tube was removed from the magnetic stand, vortexed and mixed, and then incubated at room temperature for 2 min. The solution was then collected at the bottom of the tube by brief centrifugation. Finally, the PCR tube was placed on the magnetic stand and stood for 2 min until the liquid was clear and the magnetic beads were fully absorbed. 21 μL of the liquid was removed to a new PCR tube, and the magnetic beads were not removed, to obtain the purified ligation product.

[0053] 1.5.4 Library PCR amplification First, the library PCR reaction system was prepared according to Table 9, and the reaction solution was mixed by gently blowing or tapping the tube wall with a pipette (not vortexed), and then centrifuged briefly to collect the reaction solution at the bottom of the tube. The tube was then placed on ice for use. Then, the PCR reaction program was set according to Table 10, and after the setting was completed, the prepared PCR reaction system was placed in a PCR instrument for amplification. After amplification, the library PCR amplification product was obtained and placed on ice for use.

[0054] Table 9 Library PCR amplification reaction system Note: 1) The amplification mixture was from the components in the sequencing reaction preparation kit (REF (article number): 2102, purchased from Tianjin Jinkey Medical Technology Co., Ltd.); 2) The UDB-XX PCR primer stock solution was 20 μM, which needed to be diluted to 10 μM before use; 3) The reagent configuration of this step needed to be double-checked by two persons, and the reagent configuration could be performed after the confirmation.

[0055] Table 10 PCR reaction program Note: This step needs to be reviewed by two people to confirm that the PCR program is correct. After confirming that there is no mistake, the PCR reaction can be performed.

[0056] 1.5.5 Library purification after PCR First, 45 μL of DNA purification magnetic beads are added to 50 μL of PCR reaction product, vortexed or pipetted for 10 times to mix well, incubated at room temperature for 5 min, and then centrifuged 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. Note that when aspirating the 80% ethanol, the action should be slow and the liquid should be completely removed to avoid droplets remaining on the wall of the tube. If there are a few drops of liquid remaining on the wall of the tube, centrifuge the tube briefly, place it on the magnetic stand again, and mix the liquid at the bottom of the tube with a small volume pipette. Open the cap and dry for 3-5 min until the magnetic beads are no longer shiny and have a slight crack (i.e., a slight crack is observed in the magnetic bead group). Then add 32.5 μL of Buffer EB, close the tube cap, remove the PCR tube from the magnetic stand, vortex to mix, and stand 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 and stand for about 2 min until it is fully magnetized and the liquid is clear. Aspirate 30 μL of liquid into a new 1.5 mL centrifuge tube. Do not aspirate the magnetic beads. The purified library is obtained.

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

[0058] Table 11 Library concentration quality control standards Note: A library concentration of >1 ng / μL is considered to be qualified. If it is not qualified, it needs to be re-built. If it is still not qualified after re-building, it needs to be fed back to the clinic for re-sampling.

[0059] 1.6 Library mixing Fill in the library information to be mixed into the mixing operation table (i.e., the pooling table), and calculate the sampling volume of each library and the theoretical concentration of the pooling liquid according to the library concentration and the required data volume. Arrange the libraries in the order of mixing, aspirate the corresponding volume of each library into a 1.5 mL EP tube according to the calculated mixing volume, mix well, and centrifuge briefly to collect the solution at the bottom of the tube. The library concentration is determined using a double-stranded DNA (ds-DNA) concentration determination kit (fluorescence method) and a Qubit 4.0 fluorometer, and the results are filled into the "mixed pool measured concentration" column of the mixing operation table. Compare the measured mixing concentration with the theoretical mixing concentration, and the difference between the two should be within 15%. If the difference is not within this range, the Qubit detection should be performed again.

[0060] Note: Try to control the pipetting volume of each library within 0.5-4.5 μL to ensure the sampling volume of the library as accurate as possible and not exceeding the total volume of the library.

[0061] 1.7 Preparation of DNB 1.7.1 Preparation of reagents Take out the library, TE buffer, buffer MB, enzyme MA, enzyme MB and DNB termination buffer (all from the Sequencing Reaction General Reagent Kit (Sequencing Method) DNB Preparation and Sequencing Reagent Kit, Item No.: 2010-02, Tianjin Jinkey Medical Technology Co., Ltd.) and place them on the ice box for about 0.5 h. After thawing, mix well using a vortex shaker for 5 s, centrifuge briefly and place on the ice box for standby.

[0062] 1.7.2 Preparation of DNB system 1 Take 0.2 mL octocap or PCR tube and prepare the reaction system according to the following Table 12 on ice. Mix well using 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 the following Table 13 to obtain DNB system 1.

[0063] Table 12 DNB reaction system Table 13 Reaction program 1.7.3 Preparation of DNB system 2 Take out the enzyme MB and place it on the ice box, centrifuge briefly to collect the solution at 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 at the bottom of the tube, and add the components shown in the following Table 14 on ice. Mix well using 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 the following Table 15.

[0064] Table 14 DNB reaction system Table 15 Reaction program Note: The PCR instrument needs to be preheated with a hot lid to ensure that the hot lid is at working temperature during the DNB reaction; the hot lid temperature is recommended to be set at 35°C, or as low as possible to be close to 35°C.

[0065] After the reaction is completed, immediately place it in an ice box and slowly add 20 µL of DNB stop buffer (about 1 drop) using a wide-mouth pipette tip. Adjust the pipette to 100 µL and slowly aspirate all the liquid with a wide-mouth pipette tip. Then add dropwise to mix until the liquid in the pipette tip is drained. Repeat 5-8 times. It can be stored at 4°C until used within 24 hours.

[0066] Measure the concentration using the single-stranded DNA (ssDNA) concentration assay kit (fluorescence) and a Qubit 4.0 fluorometer. A concentration >8 ng / µL is considered acceptable. If not, re-prepare DNB.

[0067] Note: a) Because DNB is relatively viscous, it is recommended to sample 2 μL for testing. For large samples, batch quantification is recommended to avoid inaccurate DNB concentration quantification due to fluorescence quenching. b) If the concentration exceeds 40 ng / µL, dilute to approximately 20 ng / µL with DNB Loading Buffer I before use.

[0068] 1.8 Sequencing 1.8.1 Reagent Preparation Remove the slides from the refrigerator and from the packaging, but leave them in the vacuum bag. Allow the slides to stand at room temperature for at least 60 minutes (no more than 24 hours). Open the vacuum bag again before use and begin DNB loading using the loading system shown in Table 16. Mix the DNB loading system gently 5-8 times with a wide-mouth pipette tip.

[0069] Table 16 DNB loading system Note: In the table: a) DNB Loading Buffer II is from the Universal Sequencing Reaction Kit (REF: 2010-02; purchased from Tianjin Jinchi Medical Technology Co., Ltd.); b) If the slide is taken out of the refrigerator and placed at room temperature and cannot be used within 24 hours, and the vacuum packaging bag is intact, it can be returned to 2-8°C for storage, but the switching between 2-8°C and room temperature should not exceed three times; c) After the vacuum packaging bag is opened, it cannot be used immediately. It can be stored at room temperature and used within 24 hours. If it exceeds 24 hours, it is not recommended to use it.

[0070] Reagents are thawed at room temperature for 3-4 hours, and placed in a 4°C refrigerator for standby. The chip needs to be balanced at room temperature for 30 minutes; the reagent needs to be inverted and mixed for 3 times before use, then the reagent tank is placed in front, and shaken left and right for 10-20 times. Open the reagent tank cover, and wipe the condensate with a dust-free paper. Take out the dNTPs mixture III and dNTPs mixture II 1 hour in advance, and melt at room temperature, then place on ice or at 4°C for standby; shake and mix for 5 seconds before use, and use after a brief centrifugation. Take out the DNA polymerase mixture before use, and place on ice or at 4°C for standby; invert and mix for 4-6 times before use. Use a clean 1 mL syringe to gently prick a 0.5 cm diameter sample hole at the edge of the No. 1 and No. 2 holes. Take the corresponding range of pipettes, and add the corresponding reagents to the corresponding holes according to the volume shown in Table 17 below. Then place the reagent tank horizontally on the table, hold both sides with both hands, shake clockwise for 10-20 times, and then counterclockwise for 10-20 times to ensure thorough mixing of the reagents. At this time, the preparation of the sequencing reagent tank before machine is completed.

[0071] Table 17 Reagent sample amount Finally, the machine is operated 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 Table 19 and Figures 2-3 Table 19 Typing detection results As can be seen from Table 19 and Figures 4-5 , the typing results of the two strains to be tested detected by the above method are: NJGLYY_14991: 369 type; NJGLYY_15736: 1728 type. The total detection time is 18 hours.

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

[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 Acinetobacter baumannii strains obtained by whole genome sequencing are as follows: NJGLYY_14991 belongs to ST369 type; and NJGLYY_15376 belongs to ST1728 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 merely describes preferred embodiments of the present application and is not intended to limit the present application in any form. Although the present application has been disclosed as above with reference to the preferred embodiments, the present application is not intended to be limited thereto. Any person skilled in the art can make some changes or modifications to the above disclosed methods and technical contents without departing from 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 still belong to the scope of the technical solution of the present application.​

Claims

1. A primer set for MLST typing of Acinetobacter baumannii based on a hospital-based microbial mNGS platform, characterized in that: Including amplification gyrB Primer pairs for amplifying gpi genes, primer pairs for amplifying gpi genes, gDJ Primer pairs for gene amplification recA Primer pairs for gene amplification cpn60 Primer pairs for gene amplification gltA The primer pairs for amplifying the gene and the primer pairs for amplifying the rpoD gene; The amplification gyrB The primer pair for the gene includes a forward primer Abaumannii_1.gyrB-F having a nucleotide sequence as shown in SEQ ID NO: 1, and a reverse primer Abaumannii_1.gyrB-R having a nucleotide sequence as shown in SEQ ID NO: 2; The amplification gpi The primer pair for the gene includes a forward primer Abaumannii_2.gpi-F having a nucleotide sequence as shown in SEQ ID NO: 3, and a reverse primer Abaumannii_2.gpi-R having a nucleotide sequence as shown in SEQ ID NO: 4; The amplification gDJ The primer pair for the gene includes a forward primer Abaumannii_3.gdhB-F having a nucleotide sequence as shown in SEQ ID NO: 5, and a reverse primer Abaumannii_3.gdhB-R having a nucleotide sequence as shown in SEQ ID NO: 6; The amplification recA The primer pair for the gene includes a forward primer Abaumannii_4.recA-F having a nucleotide sequence as shown in SEQ ID NO: 7, and a reverse primer Abaumannii_4.recA-R having a nucleotide sequence as shown in SEQ ID NO: 8; The amplification cpn60 The primer pair for the gene includes a forward primer Abaumannii_5.cpn60-F having a nucleotide sequence as shown in SEQ ID NO: 9, and a reverse primer Abaumannii_5.cpn60-R having a nucleotide sequence as shown in SEQ ID NO: 10; The amplification gltA The primer pair for the gene includes a forward primer Abaumannii_6.gltA-F having a nucleotide sequence as shown in SEQ ID NO: 11, and a reverse primer Abaumannii_6.gltA-R having a nucleotide sequence as shown in SEQ ID NO: 12; and The amplification rpoD The primer pair for the gene includes a forward primer Abaumannii_7.rpoD-F having a nucleotide sequence as shown in SEQ ID NO: 13, and a reverse primer Abaumannii_7.rpoD-R having a nucleotide sequence as shown in SEQ ID NO:

14.

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

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

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

5. A mNGS sequencing library construction method for MLST typing of Acinetobacter baumannii, 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 test 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 Acinetobacter baumannii.

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