Targeted sequencing primer group and kit for detecting common pathogens of children and drug-resistant genes thereof

Abstract

The invention belongs to the technical field of biological detection, and particularly discloses a targeted sequencing primer group and a kit for detecting common pathogens of children and drug-resistant genes thereof. The primer group comprises 145 primer pairs of common children pathogen primer groups and 67 primer pairs of drug-resistant gene primer groups of the common children pathogen primer groups. According to the kit provided by the invention, nucleic acid in a clinical sample is directly extracted, 212 pairs of specific primers are adopted to realize multi-targeted amplification of a pathogen target area, a high-throughput sequencing platform is adopted to accurately identify pathogens and drug-resistant mutant genes thereof, 72 common children pathogens and drug-resistant genes thereof can be detected at the same time, and the kit has a wide application prospect. More accurate diagnosis information is provided for clinic, a key technical support is provided for early-stage accurate treatment of children infectious diseases, and the technical problems that in the prior art, in children pathogen detection, the detection speed is low, the number of missed detection is large, and the drug-resistant blind area is large are solved.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological detection, and particularly relates to a metagenomic primer set and kit for detecting common pathogenic bacteria of children and drug-resistant genes thereof. BACKGROUND

[0002] Children's immune systems are not yet fully developed, so infectious diseases are common in children. These diseases are usually caused by various pathogens such as bacteria, viruses, fungi, etc., and have characteristics such as acute onset and non-specific clinical symptoms. The high incidence and high mortality rate of infectious diseases in children constitute an important challenge to global public health. According to the data in 2019, more than 300 million children under the age of 5 died of infectious diseases, accounting for more than 50% of the number of children under the age of 5. From the perspective of clinical microbiology and epidemiology, the distribution of pathogens of infectious diseases in children shows significant anatomical site specificity, age dependence and regional heterogeneity and seasonal fluctuation.

[0003] In recent years, due to the problems of pathogen evolution accelerated by horizontal gene transfer and spontaneous mutation, and the abuse of antibacterial drugs in pediatrics, the drug resistance of infectious pathogens in children has become increasingly serious, and has become a major challenge in the field of global public health. Epidemiological surveillance data shows that the drug resistance rate of major pathogens is showing a continuous upward trend: the drug resistance rate of Streptococcus pneumoniae to penicillin has reached 35.6%, and the proportion of methicillin-resistant Staphylococcus aureus in pediatric isolates is more than 42.8%, with an annual growth rate of 6.9%. This drug resistance crisis has led to a 28% increase in the failure rate of traditional empirical treatment, significantly prolonging hospitalization time and increasing medical costs. Under this background, the development of pathogen-drug gene joint detection technology with high sensitivity and specificity has become a key breakthrough to improve the prognosis of infectious diseases in children and reduce the mortality rate of such diseases.

[0004] Targeted next-generation sequencing technology (tNGS) provides a new solution for detecting common infectious pathogens in children due to its high throughput, high sensitivity, and high resolution. tNGS technology designs a large number of primers or probes targeting specific pathogen gene sequences, performs multiplex PCR amplification or probe capture on nucleic acids in the sample to be tested, thereby obtaining a large number of target nucleic acid fragments, and then sequencing these fragments through high-throughput sequencing technology, and finally combining bioinformatics analysis to achieve high sensitivity and high accuracy identification of pathogens in the sample to be tested. For example, CN117757962A provides a tNGS kit and method for detecting multiple pathogens, which can simultaneously detect 96 infectious pathogens including bacteria, mycobacteria, viruses, and fungi. CN118813840A provides a targeted metagenomic primer set and kit for respiratory tract pathogens, which can cover a variety of respiratory pathogens, including 3 gram-positive bacteria, 6 gram-negative bacteria, 1 mycoplasma, 7 DNA viruses, 5 RNA viruses, and 5 fungi. However, the above technologies are mainly for adult pathogen detection, and due to the huge difference in pathogen spectrum between children and adults, the detection efficiency is often low, which cannot meet the detection needs of infectious diseases in children.

[0005] Therefore, designing and developing a targeted metagenomic primer set and kit for common pathogens and drug-resistant genes in children can effectively make up for the shortcomings of existing technologies, improve the accuracy and sensitivity of detection by targeting the unique pathogen spectrum characteristics of children, and provide more accurate diagnostic information for clinical practice, making early and accurate treatment possible, and providing key technical support for improving the prognosis of infectious diseases in children. SUMMARY

[0006] To solve the above technical problems, the present application designs a targeted metagenomic primer set based on the characteristics of common pathogens in children and their drug-resistant gene characteristics, and develops a kit for detecting common pathogens in children and their drug-resistant genes on this basis, which can simultaneously detect 72 common pathogens in children and their drug-resistant genes, thereby providing more accurate diagnostic information for clinical practice, making early and accurate treatment possible, and providing key technical support for improving the prognosis of infectious diseases in children.

[0007] In one aspect, the present application provides a primer set, which is composed of a common pathogen primer set for children and a drug-resistant gene primer set for children;

[0008] The common pathogen primer set for children is composed of 145 pairs of primer pairs, each pair of primer pairs is composed of a forward primer and a reverse primer, and the primer pairs are selected from SEQ ID NO: 1-290;

[0009] The drug resistance gene primer set consists of 67 pairs of primer pairs, each pair of primer pair consisting of a forward primer and a reverse primer, the primer pair being selected from SEQ ID NO: 291-424.

[0010] In a second aspect, the present application also provides a kit for detecting common pathogenic bacteria and drug resistance genes of children, the kit being used for detecting common pathogenic bacteria and drug resistance genes of children;

[0011] The common pathogenic bacteria of children are at least one of Enterococcus faecalis, Enterococcus faecium, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus agalactiae, Streptococcus pneumoniae, Listeria monocytogenes, Mycobacterium tuberculosis complex, Escherichia coli, Haemophilus influenzae, Klebsiella pneumoniae, Moraxella catarrhalis, Pseudomonas aeruginosa, Stenotrophomonas maltophilia, Enterobacter cloacae complex, Acinetobacter baumannii, Bacteroides fragilis, Bordetella pertussis, Legionella pneumophila, Mycoplasma pneumoniae, Chlamydia trachomatis, Human adenovirus A, Human adenovirus B, Human adenovirus C, Human adenovirus D, Human adenovirus E, Human adenovirus F, Human adenovirus G, Human herpesvirus 1, Human herpesvirus 2, Varicella-zoster virus, Human herpesvirus 4, Human cytomegalovirus, Human herpesvirus 6, Human bocavirus, Human polyomavirus 1, Human polyomavirus 2, Primate erythrovirus 1, Rhinovirus A, Rhinovirus B, Rhinovirus C, Respiratory syncytial virus A, Respiratory syncytial virus B, Human metapneumovirus, Parainfluenza virus 1, Parainfluenza virus 3, Influenza A virus, Influenza A virus H1N1, Influenza A virus H3N2, Influenza A virus H5N1, Influenza A virus H7N9, Influenza B virus, Novel coronavirus, Rotavirus, Rotavirus A, Norovirus, Enterovirus A, Enterovirus B, Enterovirus C, Enterovirus D, Parechovirus A, Parechovirus B, Parechovirus C, Parechovirus D, Enterovirus A71, Echovirus 11, Aspergillus flavus, Aspergillus fumigatus, Candida albicans, Candida parapsilosis, Candida tropicalis, Pneumocystis jirovecii;

[0012] The drug resistance gene is at least one of CTX-M, SHV, TEM, KPC, NDM, VIM, IMP, OXA-48, OXA-23, mecA, vanA, vanB, mcr-1, 23S rRNA-A2063G, 23S rRNA-A2064G, 23S rRNA-A2067G, 23S rRNA-C2617G;

[0013] The common pathogenic bacteria primer set and the drug resistance gene primer set of the present application are selected according to the composition of the common pathogenic bacteria of children.

[0014] Further, the kit comprises a nucleic acid extraction kit, a reverse transcription kit, the primer set of the application, a multiplex PCR reagent, a purification kit, and a sequencing reaction universal reagent.

[0015] Further, in the kit, the nucleic acid extraction kit is a magnetic bead method pathogenic microorganism DNA / RNA extraction kit.

[0016] Further, in the kit, the multiplex PCR reagent comprises a premix reagent for simultaneously amplifying DNA and reverse transcription products.

[0017] Further, in the kit, the purification kit comprises purification magnetic beads and an enzyme digestion premix.

[0018] In a third aspect, the application further provides a method for detecting a plurality of common pathogenic microorganisms of children and drug resistance genes thereof based on metagenomics, which is a non-disease diagnosis method, comprising: collecting a sample, extracting pathogenic microorganism nucleic acids in the sample using a nucleic acid extraction kit;

[0019] establishing a multiplex PCR reaction system, amplifying using the kit for detecting common pathogenic microorganisms of children and drug resistance genes thereof provided by the application; purifying the amplification products; library quality control, mixing the qualified library for sequencing; based on the sequencing results, determining the common pathogenic microorganisms of children and drug resistance genes thereof in the sample.

[0020] Further, in the detection method, the sample is one or more of lung lavage fluid, blood, or cerebrospinal fluid of a child patient.

[0021] Further, in the detection method, the library standard for quality control is a library concentration > 1 ng / μL.

[0022] Further, in the detection method, in the library mixing for sequencing, the fragment size of the library with different indexes is 350 bp.

[0023] Compared with the prior art, the technical solution provided by the application has at least the following beneficial effects or advantages:

[0024] The application discloses a metagenomic primer set and kit for detecting common pathogenic microorganisms of children and drug resistance genes thereof, and uses targeted metagenomics to detect common pathogenic microorganisms of children and drug resistance genes thereof.

[0025] The present application directly extracts nucleic acid in a clinical sample, uses 212 pairs of specific primers to realize multiplex target amplification of pathogen target regions, uses a high-throughput sequencing platform to accurately identify pathogens and their drug-resistant mutations, and reduces the risk of misdiagnosis and missed diagnosis. Assist the clinic in rapid and accurate anti-infection treatment. At the same time, the pathogen types covered by the kit are relatively complete, the operation is relatively simple, and the detection is rapid and accurate. For qualitative detection of common pathogenic bacteria and their drug-resistant genes in children, only a small amount of clinical sample is needed, and the synchronous detection of 72 kinds of pathogenic bacteria and their drug-resistant genes can be completed at one time, and the whole process operation can be realized within 20h-24h. Coupled with the data analysis system, the detection results can be automatically output, reducing human operation errors, improving the accuracy and efficiency of data interpretation, and further simplifying the burden of clinical work.

[0026] In summary, the macrogenomic primer set and kit for detecting common pathogenic bacteria and their drug-resistant genes in children provided by the present application solve the technical problems of slow detection speed, missed detection, and large drug resistance blind area for detecting pathogenic bacteria in children, have the advantages of high sensitivity, rapidness, and economy, and have extremely high clinical application value. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 The kit detection flowchart is shown. DETAILED DESCRIPTION

[0028] In the following, the technical solutions of the present application will be described in conjunction with examples, but the present application is not limited to the following examples.

[0029] In order to enable those skilled in the art to better understand the technical solutions of the present application and to implement them, the present application will be further described in conjunction with specific examples and drawings, but the examples are not limiting to the present application.

[0030] The experimental methods and detection methods described in the following examples are all conventional methods unless otherwise specified; the reagents and materials described are all commercially available unless otherwise specified.

[0031] Example 1

[0032] This example is intended to illustrate the use of the macrogenomic primer set and kit for detecting common pathogenic bacteria and their drug-resistant genes in children provided by the present application.

[0033] For detecting common pathogenic bacteria and their drug-resistant genes in children, as shown in the following steps: Figure 1

[0034] (1) Sample DNA / RNA nucleic acid extraction: extracting nucleic acid from the lung lavage fluid, blood and cerebrospinal fluid or other samples of the child;

[0035] ​(2) Reverse transcription: reverse transcription of RNA in the DNA / RNA extracted in (1) using a reverse transcription reagent;

[0036] (3) Multiplex PCR targeted amplification: first round of PCR amplification of the reverse transcription product containing the DNA in (1) obtained in (2) using multiplex PCR targeted primers;

[0037] (4) PCR product purification: purification of the first round of PCR product obtained in (3) to obtain a first round of PCR purified product;

[0038] (5) Library amplification: second round of PCR amplification using the first round of PCR purified product obtained in (4) as a template while adding known index and universal adapter sequences to complete library amplification;

[0039] (6) Library purification + pooling: purification and pooling of the library obtained in (5);

[0040] (7) Library sequencing;

[0041] (8) Data analysis and report generation: analysis of the sequencing results by bioinformatics and generation of a report.

[0042] The multiplex PCR targeted primers provided in the embodiment comprise 72 pathogen-specific primers and 17 drug resistance gene-specific primers, the sequence information of the 72 pathogen-specific primers is shown in Table 1, and the sequence information of the 17 drug resistance gene-specific primers is shown in Table 2. The 17 drug resistance genes are: CTX-M, SHV, TEM, KPC, NDM, VIM, IMP, OXA-48, OXA-23, mecA, vanA, vanB, mcr-1, 23S rRNA-A2063G, 23S rRNA-A2064G, 23S rRNA-A2067G, and 23S rRNA-C2617G. The 72 pathogenic bacteria comprise 8 Gram-positive bacteria, 10 Gram-negative bacteria, 3 special pathogens, 17 DNA viruses, 28 RNA viruses, and 6 fungi. The specific pathogenic bacteria involved are as follows:

[0043] 8 Gram-positive bacteria: Enterococcus faecalis, Enterococcus faecium, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus agalactiae, Streptococcus pneumoniae, Listeria monocytogenes, Mycobacterium tuberculosis complex.

[0044] 10 Gram-negative bacteria: Escherichia coli, Haemophilus influenzae, Klebsiella pneumoniae, Moraxella catarrhalis, Pseudomonas aeruginosa, Stenotrophomonas maltophilia, Enterobacter cloacae complex, Acinetobacter baumannii, Bacteroides fragilis, Bordetella pertussis; 3 special pathogens: Legionella pneumophila, Mycoplasma pneumoniae, Chlamydia trachomatis.

[0045] 17 DNA viruses: human adenovirus A, human adenovirus B, human adenovirus C, human adenovirus D, human adenovirus E, human adenovirus F, human adenovirus G, human herpesvirus 1 (HSV1), human herpesvirus 2 (HSV2), varicella-zoster virus (VZV), human herpesvirus 4 (EBV), human cytomegalovirus (CMV), human herpesvirus 6, human bocavirus, human polyomavirus 1 (BK virus), human polyomavirus 2 (JC virus), primate erythrovirus 1.

[0046] 28 RNA viruses: rhinovirus A, rhinovirus B, rhinovirus C, respiratory syncytial virus A, respiratory syncytial virus B, human metapneumovirus, parainfluenza virus 1, parainfluenza virus 3, influenza A virus, influenza A virus H1N1, influenza A virus H3N2, influenza A virus H5N1, influenza A virus H7N9, influenza B virus, novel coronavirus, rotavirus, rotavirus type A, norovirus, enterovirus A, enterovirus B, enterovirus C, enterovirus D, coxsackievirus A type, coxsackievirus B type, coxsackievirus C type, coxsackievirus D type, enterovirus A71, echovirus 11.

[0047] 6 fungi: Aspergillus flavus, Aspergillus fumigatus, Candida albicans, Candida parapsilosis, Candida tropicalis, Pneumocystis jirovecii.

[0048] Table 1. 72 pathogen primer sequence information table

[0049]

[0050]

[0051]

[0052]

[0053]

[0054]

[0055]

[0056]

[0057] Table 2. 17 drug-resistant gene primer sequence information table

[0058]

[0059]

[0060]

[0061]

[0062] The embodiment can detect the above-mentioned 17 drug resistance genes and 72 pathogenic bacteria or one or several thereof according to the actual clinical needs, and generate a report according to the detection results for clinical medication guidance.

[0063] Example 2

[0064] The embodiment is for detection of clinical samples of children.

[0065] 2.1 Collecting patient samples and performing sample pretreatment

[0066] Collecting alveolar lavage fluid, blood or cerebrospinal fluid samples of patients in clinic, and pretreating samples in different ways according to sample types.

[0067] Alveolar lavage fluid: if the alveolar lavage fluid is colorless and transparent liquid, directly take 200 μL for nucleic acid extraction; if the alveolar lavage fluid is non-transparent liquid, add appropriate amount of PBS for dilution until the alveolar lavage fluid is not sticky, centrifuge 1600g of the above-mentioned non-transparent alveolar lavage fluid diluent for 5 min, and take 200 μL supernatant for nucleic acid extraction.

[0068] Blood: centrifuge 1600g for 5 min, and take 200 μL supernatant for nucleic acid extraction.

[0069] Cerebrospinal fluid: directly take 200 μL for nucleic acid extraction.

[0070] 2.2 Nucleic acid extraction of clinical samples

[0071] Using the magnetic bead method pathogenic microorganism DNA / RNA extraction kit to extract nucleic acid in samples, the operation is as follows:

[0072] (1) Take 1 tube of 200 μL RNase-free water, and add 150 copies of internal reference as negative quality control.

[0073] (2) Take enough 1.5 mL centrifuge tube, number, and add 200 μL of treated sample to be tested according to the order, and add 150 copies of internal reference.

[0074] (3) Add 20 μL of nucleic acid protection solution and 20 μL of proteinase K to the centrifuge tube, add 600 μL of lysis and binding liquid, and shake well for 30 s.

[0075] (4) Add 20 μL of magnetic beads, shake at high speed for 30 s, then mix well with 3D mixer for 4 min, and shake well for 30 s again, so that the magnetic beads adsorb the nucleic acid, and then take off the instantaneous separation.

[0076] (5) Transfer to the magnetic stand for magnetic separation until the solution is clear. It takes at least 3 minutes for blood and other invisible magnetic beads to be adsorbed, and then the solution is discarded.

[0077] (6) Add 700 μL of washing solution 1, vortex for 30 s, then transfer to the magnetic stand for adsorption until the solution is clear. Discard the solution.

[0078] (7) Add 700 μL of washing solution 2, vortex for 30 s to disperse the magnetic beads, then transfer to the magnetic stand for magnetic separation until the solution is clear. Discard the solution.

[0079] (8) Add 700 μL of washing solution 2 again, vortex for 30 s to disperse the magnetic beads, then transfer to the magnetic stand for magnetic separation until the solution is clear. Discard the solution.

[0080] (9) After brief centrifugation, transfer to the magnetic stand for adsorption. After adsorption, discard the supernatant with a 100 μL pipette.

[0081] (10) Add 50 μL of nuclease-free water, shake well for 30 s, and centrifuge.

[0082] (11) Transfer to the magnetic stand for adsorption. After adsorption, take 48 μL of the supernatant to a new 1.5 mL centrifuge tube to prepare the nucleic acid, and place the prepared nucleic acid in an ice box in time.

[0083] 2.3 Nucleic acid quality control

[0084] The extracted DNA is detected for concentration using the dsDNA HS Assay Kit. A concentration of >0 ng / μL is considered acceptable.

[0085] 2.4 Reverse transcription

[0086] (1) Take the reverse transcription kit HiScript III 1st Strand cDNA Synthesis Kit and the DNA / RNA sample prepared in 2.2 from the ice box, melt, shake slightly, centrifuge, and place on the ice box to prepare the RNA in the sample for reverse transcription. The DNA in the sample does not participate in the reverse transcription reaction. The reverse transcription reaction system is as follows:

[0087]

[0088] (2) Carefully pipette 2.5 μL of the reverse transcription mixture into the prepared 0.2 mL PCR tube.

[0089] (3) According to the corresponding number order, sequentially add 8.5 μL of the sample (the total amount of nucleic acid in the sample does not exceed 2 μg) to the PCR tube containing the Mix, shake well, and centrifuge.

[0090] PCR tube is placed on the PCR instrument, run the following program (20 μL system), hot cover 105℃, PCR reaction program as follows:

[0091]

[0092] Program end to take out the sample containing the reverse transcription product of DNA not involved in reverse transcription, can immediately proceed to the next step or-20℃ temporary storage, to avoid repeated freeze-thaw.

[0093] 2.5 First round of multiplex PCR amplification

[0094] DNA & RNA Multiplex PCR Mix, 2.4 obtained from the reverse transcription product containing DNA samples not involved in reverse transcription corresponding primers, melt slightly shaken, centrifuged, placed on the ice box for standby. According to the following system to prepare the first round of PCR reaction premix:

[0095]

[0096] 11 μL 2.4 obtained from the reverse transcription product containing DNA samples not involved in reverse transcription and 9 μL premix mixed into 0.2 mL PCR tube, slight vibration, centrifuged. According to the following PCR reaction program for the first round of PCR amplification:

[0097] First round of multiplex PCR reaction program

[0098]

[0099]

[0100] 2.6 First round of PCR product purification and second round of PCR amplification

[0101] (1) Prepare magnetic beads in advance, room temperature equilibrium for more than 30 min

[0102] (2) configuration of the second round of PCR amplification premix:

[0103] ① from-20℃ take out 2×Taq PCR Mastermix, dUTP (10 mM), Index, room temperature melting, slightly shaken, centrifuged, placed on the ice box for standby.

[0104] ② according to the experimental requirements of the arrangement of Index sequence, according to the following system to prepare the second round of PCR reaction premix:

[0105]

[0106] (3) according to the following system configuration of the first round of PCR product purification magnetic beads:

[0107]

[0108] (4) The enzyme digestion premix solution is configured according to the following system:

[0109]

[0110] (5) After the first round of PCR amplification, the product is cooled in a refrigerator at -20°C for 1 min.

[0111] (6) After taking out from the refrigerator, the magnetic beads are added, vortexed, and mixed, and the reaction tube is placed on the magnetic stand after instant centrifugation.

[0112] (7) The supernatant is removed with a 200 μL gun head, and the supernatant is removed again after instant centrifugation by replacing the 10 μL gun head, to ensure that the supernatant is completely removed.

[0113] (8) The enzyme digestion premix solution (20 μL) is added to the PCR tube.

[0114] (9) The PCR tube is removed, the enzyme digestion premix solution is mixed by shaking, and the enzyme digestion is carried out in the following enzyme digestion program:

[0115] Enzyme digestion reaction program (heat cover setting 105°C, reaction volume 20 μL)

[0116]

[0117] (10) After the enzyme digestion is completed, the product is cooled in a refrigerator at -20°C for 1 min.

[0118] (11) The product obtained in (10) is taken out from the refrigerator, and the second round of PCR amplification premix solution is added, vortexed, and mixed, and the second round of PCR amplification is carried out after instant centrifugation.

[0119] (12) The following PCR reaction program is set:

[0120] Second round of PCR reaction program

[0121]

[0122] (13) After the PCR is completed, instant centrifugation is carried out.

[0123] 2.7 Second round of PCR product purification

[0124] (1) 40 μL of magnetic beads are added to the second round of PCR reaction system after the PCR is completed, vortexed, and mixed, and combined at room temperature for 5 min.

[0125] (2) The magnetic stand is placed, and the solution is clear and transparent, and the supernatant is discarded.

[0126] (3) Add 180 μL of freshly prepared 80% ethanol, rinse once and discard the supernatant.

[0127] (4) Repeat the rinse with 180 μL of 80% ethanol once and discard the supernatant.

[0128] (5) After standing, use a 10 μL pipette to aspirate the residual ethanol, and discard the residual ethanol.

[0129] (6) Add 30 μL of RNase-free water for elution, vortex well, and stand at room temperature for 5 min.

[0130] (7) After instant separation, place it in a magnetic stand, and after the magnetic beads are completely adsorbed, aspirate 27 μL of supernatant into a prepared storage tube, which completes the library construction, i.e. amplification, and prepares the library.

[0131] 2.8 Library quality control

[0132] The library prepared in 2.7 is detected for concentration using a dsDNA HS Assay Kit for DNA, and the library DNA concentration is greater than >1 ng / μL, which is qualified.

[0133] 2.9 Sequencing on machine

[0134] (1) Pooling strategy: sample library is pooled for sequencing according to an average fragment size of 350 bp;

[0135] (2) If sequencing alone, add phix (30%).

[0136] The kit of the present application (this example) and the currently widely used metagenomic sequencing (mNGS) are used as a comparative example to detect 128 different types of clinical samples, including 75 mNGS positive alveolar lavage fluid samples and 6 mNGS negative alveolar lavage fluid samples; 29 mNGS positive blood samples and 4 mNGS negative blood samples; 10 mNGS positive cerebrospinal fluid samples and 4 mNGS negative cerebrospinal fluid samples (target detection is within the coverage of the product), a total of 179 groups of mNGS report results, according to Table 1, the samples are counted, the detection results are analyzed, the detection sensitivity, specificity, positive predictive value, negative predictive value and overall coincidence rate are calculated, and the detection results are shown in Tables 2 and 3.

[0137] Table 1 Sample statistics

[0138]

[0139] TP (True Positive) in Table 1: the number of correctly detected positive samples; TN (True Negative): the number of correctly detected negative samples; FP (False Positive): the number of misjudged negative samples as positive; FN (False Negative): the number of missed positive samples. The calculation formulas of sensitivity, specificity, positive predictive value, negative predictive value and overall coincidence rate are as follows:

[0140] Sensitivity (%) = TP / (TP + FN) x 100

[0141] Specificity (%) = TN / (TN + FP) x 100

[0142] Positive predictive value (%) = TP / (TP + FP) x 100

[0143] Negative predictive value (%) = TN / (TN + FN) x 100

[0144]

[0145] Table 2: Performance evaluation results of clinical sample detection

[0146]

[0147] Table 3: Report compliance results

[0148]

[0149] As shown in the above table, the primer set and kit of the present application can better detect the common pathogen infection in children, including gram-positive bacteria, gram-negative bacteria, viruses, fungi, special pathogens and other microorganism species. The sensitivity, specificity, positive predictive value, negative predictive value and overall coincidence rate are comparable to mNGS. The results show that the macrogenomic primer set and kit for detecting common pathogen and drug resistance gene of children provided by the present application can complete the simultaneous detection of 72 pathogenic bacteria of children and the simultaneous detection of 17 drug resistance genes at one time only with a small amount of clinical sample. The whole process can be completed in 20-24h, and the data analysis system can automatically output the detection results, guide the clinical medication, improve the accuracy of medication, reduce the side effects of drugs and reduce the probability of drug-resistant bacteria.

[0150] As described above, the basic principles, main features and advantages of the present application are better described. The above examples and specification only describe the preferred embodiments of the present application, and the present application is not limited by the above examples. Without departing from the spirit and scope of the present application, various changes and improvements to the technical solutions of the present application made by those skilled in the art shall fall within the scope of protection of the present application.

Claims

1. A primer set, characterized in that: The primer set consists of a primer set for common childhood pathogens and a primer set for drug-resistant genes; The common childhood pathogen primer set consists of 145 primer pairs, each primer pair consists of a forward primer and a reverse primer, and the primer pairs are selected from SEQ ID NOs: 1 to 290; The drug-resistant gene primer set consists of 67 pairs of primer pairs, each pair of primer pairs consists of a forward primer and a reverse primer, and the primer pairs are selected from SEQ ID NOs: 291-424.

2. A kit for detecting common pathogens in children and their drug-resistant genes, characterized in that: The kit is used to detect common pathogens in children and their drug-resistant genes; The common pathogens in children are Enterococcus faecalis, Enterococcus faecium, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus agalactiae, Streptococcus pneumoniae, Listeria monocytogenes, Mycobacterium tuberculosis complex, Escherichia coli, Haemophilus influenzae, Klebsiella pneumoniae, Moraxella catarrhalis, Pseudomonas aeruginosa, Stenotrophomonas maltophilia, Enterobacter cloacae complex, Acinetobacter baumannii, Bacteroides fragilis, Bordetella pertussis, Legionella pneumophila, Mycoplasma pneumoniae, Chlamydia trachomatis, human adenovirus A, human adenovirus B, human adenovirus C, human adenovirus D, human adenovirus E, human adenovirus F, human adenovirus G, human herpes virus type 1, human herpes virus type 2, varicella zoster virus, human herpes virus type 4, human cytomegalovirus, human herpes virus 6, human bocavirus, human polyomavirus type 1, human At least one of polyomavirus type 2, primate erythrocytic parvovirus 1, rhinovirus A, rhinovirus B, rhinovirus C, respiratory syncytial virus A, respiratory syncytial virus B, human metapneumovirus, parainfluenza virus 1, parainfluenza virus 3, influenza A virus, influenza A virus H1N1, influenza A virus H3N2, influenza A virus H5N1, influenza A virus H7N9, influenza virus B, novel coronavirus, rotavirus, rotavirus type A, norovirus, enterovirus A, enterovirus B, enterovirus C, enterovirus D, double echovirus type A, double echovirus type B, double echovirus type C, double echovirus type D, enterovirus A71, echovirus 11, Aspergillus fumigatus, Aspergillus albicans, Candida parapsilosis, Candida tropicalis, and Pneumocystis jiroveci; The drug-resistant gene is at least one of CTX-M, SHV, TEM, KPC, NDM, VIM, IMP, OXA-48, OXA-23, mecA, vanA, vanB, mcr-1, 23S rRNA-A2063G, 23S rRNA-A2064G, 23S rRNA-A2067G, and 23S rRNA-C2617G; The common childhood pathogen primer set and drug resistance gene primer set of claim 1 are selected according to the composition of the common childhood pathogens.

3. The kit according to claim 2, wherein The invention comprises a nucleic acid extraction kit, a reverse transcription kit, the primer set according to claim 1, a multiplex PCR kit, a purification reagent, and a universal reagent for sequencing reaction.

4. The kit according to claim 3, wherein The nucleic acid extraction kit is a magnetic bead method pathogenic microorganism DNA / RNA extraction kit.

5. The kit according to claim 3, characterized in that The multiplex PCR reagents include premixed reagents for simultaneously amplifying DNA and reverse transcription products.

6. The kit according to claim 3, characterized in that The purification kit comprises purification magnetic beads and enzyme digestion premix.

7. A metagenomics-based method for detecting multiple common childhood pathogens and their drug-resistant genes, which is a non-disease diagnostic method, characterized in that: include: Collect samples and use a nucleic acid extraction kit to extract pathogen nucleic acid from the samples; Establish a multiplex PCR reaction system, use the kit for detecting common childhood pathogens and their drug-resistant genes described in claim 2 for amplification; purify the amplified products; perform library quality control, and mix the quality-controlled qualified libraries for sequencing; based on the sequencing results, determine the common childhood pathogens and their drug-resistant genes in the sample.

8. The detection method according to claim 7, characterized in that The sample is one or more of the patient's alveolar lavage fluid, blood or cerebrospinal fluid.

9. The detection method according to claim 7, characterized in that The quality control qualified library standard is library concentration>1ng / μL.

10. The detection method according to claim 7, characterized in that: In the mixed library sequencing, the size of the fragments of the libraries with different indexes was 350 bp.

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