Identification of common respiratory pathogens in children based on nucleic acid mass spectrometry

Abstract

The application discloses a nucleic acid mass spectrometry technology-based method for identifying common respiratory tract infection pathogens in children. The application provides a complete set of primers, including specific primer group 1 and specific primer group 2; the primer group 1 (i.e. the primer corresponding to well1) is composed of single-stranded DNA molecules shown in sequences 1-17 and single-stranded DNA molecules shown in sequences 24-40; the primer group 2 (i.e. the primer corresponding to well2) is composed of single-stranded DNA molecules shown in sequences 18-23 and single-stranded DNA molecules shown in sequences 41-46. The CCRP-MS of the application is a 23-pathogen determination method combining matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and multiple reverse transcription polymerase chain reaction (MRT-PCR), and can directly detect common respiratory tract pathogens in children.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and relates to a method for identifying common respiratory infection pathogens in children based on nucleic acid mass spectrometry. BACKGROUND

[0002] Acute respiratory tract infections (ARTIs) are mainly related to the upper respiratory tract and / or lower respiratory tract, accounting for most of the infectious diseases in the world. ARTIs can be caused by a variety of pathogens, such as bacteria, viruses, mycoplasma, chlamydia, etc. Each year, millions of people are hospitalized and die due to ARTIs. ARTIs are one of the main causes of morbidity and mortality in children under the age of five worldwide, causing 4 million deaths each year (Li et al., 2019; Kwon et al., 2021; Katherine et al., 2021). Many pathogens can cause ARTIs, but the symptoms and signs mediated by different pathogens are relatively similar, and it is difficult to distinguish them (Wu et al., 2017), which brings great challenges to clinical diagnosis and treatment. Therefore, it is crucial to identify the pathogen early for clinical diagnosis and treatment.

[0003] Currently, the traditional pathogen detection methods are mainly culture method and serological detection method. These methods have a longer processing period (3 to 5 days), a more complex process and a low sensitivity. Some molecular biology methods such as PCR can quickly and sensitively diagnose pathogens. However, the throughput of these reactions is not high enough. Although there are now many advanced methods such as gene chips and high-throughput sequencing technology, the high cost hinders their widespread use (Cox et al., 2019).

[0004] With the great progress of matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) technology, this technology is now increasingly used in precision medicine and public health (Magda et al., 2021). This method is known for its high sensitivity, high accuracy, high throughput, low cost and detection of up to 30 or more pathogens. It has been widely used in pathogen detection, such as detection of 21 common respiratory viruses (Zhang et al., 2015), detection of pneumonia and meningitis (Zhang et al., 2018), detection of 10 duck viruses (Ning et al., 2019), detection of SARS-CoV-2 variants (Zhao et al., 2021), etc. SUMMARY

[0005] The purpose of the present application is to provide a method for identifying common respiratory infection pathogens in children based on nucleic acid mass spectrometry.

[0006] In a first aspect, the present application provides a set of primers comprising a specific primer group;

[0007] The specific primer set consists of single-stranded DNA molecules shown in SEQ ID NO: 1-16, single-stranded DNA molecules shown in SEQ ID NO: 24-39, single-stranded DNA molecules shown in SEQ ID NO: 18-22, and single-stranded DNA molecules shown in SEQ ID NO: 41-45.

[0008] The specific primer set described above consists of specific primer set 1 and specific primer set 2;

[0009] The specific primer set 1 (i.e. the primer corresponding to well 1) consists of single-stranded DNA molecules shown in SEQ ID NO: 1-16 and single-stranded DNA molecules shown in SEQ ID NO: 24-39;

[0010] The specific primer set 2 (i.e. the primer corresponding to well 2) consists of single-stranded DNA molecules shown in SEQ ID NO: 18-22 and single-stranded DNA molecules shown in SEQ ID NO: 41-45.

[0011] The specific primer set 1 and the specific primer set 2 described above are packaged independently.

[0012] The kit of primers described above further comprises a single-base extension primer set;

[0013] The single-base extension primer set consists of single-stranded DNA molecules shown in SEQ ID NO: 47-62 and single-stranded DNA molecules shown in SEQ ID NO: 64-68.

[0014] The single-base extension primer set described above consists of single-base extension primer set 1 and single-base extension primer set 2;

[0015] The single-base extension primer set 1 (i.e. the single-base extension primer set corresponding to well 1) consists of single-stranded DNA molecules shown in SEQ ID NO: 47-62;

[0016] The single-base extension primer set 2 (i.e. the single-base extension primer set corresponding to well 2) consists of single-stranded DNA molecules shown in SEQ ID NO: 64-68.

[0017] The single-base extension primer set 1 and the single-base extension primer set 2 described above are packaged independently.

[0018] In a second aspect, the present application provides MRT-PCR reagents, which are any one of, or A1) and A2), or A3) and A4), or A1)-A4):

[0019] A1) MRT-PCR reagent 1 containing the specific primer set 1 in the first aspect;

[0020] A2) MRT-PCR reagent 2 containing the specific primer set 2 in the first aspect;

[0021] A3) MRT-PCR reagent 3 containing the single base extension primer set 1 of the first aspect;

[0022] A4) MRT-PCR reagent 4 containing the single base extension primer set 2 of the first aspect.

[0023] In a third aspect, the present application provides the use of the primer set of the first aspect, or the MRT-PCR reagent of the second aspect in any one of the following:

[0024] C1) preparing a kit for detecting or aiding the detection of a common childhood respiratory pathogen or nucleic acid thereof;

[0025] C2) preparing a kit for screening or aiding the screening of a common childhood respiratory pathogen or nucleic acid thereof;

[0026] C3) preparing a kit for identifying or aiding the identification of whether a common childhood respiratory pathogen or nucleic acid thereof is present in a sample to be tested;

[0027] C4) preparing a kit for identifying or aiding the identification of whether a common childhood respiratory pathogen or nucleic acid thereof is present in a sample to be tested.

[0028] In a fourth aspect, the present application provides a kit comprising the following components:

[0029] B1) the primer set of the first aspect;

[0030] B2) the MRT-PCR reagent of the second aspect.

[0031] The above-mentioned kit further comprises the following:

[0032] B3) shrimp alkaline phosphatase;

[0033] B4) instruments or reagents required for nucleic acid mass spectrometry detection.

[0034] In the above-mentioned kit, in addition to the primer sequences of B1) and B2), the instruments or reagents required for nucleic acid mass spectrometry detection, or other reagents used for amplification or shrimp alkaline phosphatase digestion are all from the time-of-flight mass spectrometry nucleic acid detection universal kit (RNA).

[0035] In a fifth aspect, the present application provides the use of the kit of the fourth aspect in any one of the following:

[0036] D1) preparing a product for detecting or aiding the detection of a common childhood respiratory pathogen or nucleic acid thereof;

[0037] D2) preparing a product for screening or aiding the screening of a common childhood respiratory pathogen or nucleic acid thereof;

[0038] D3) preparing to identify or assist in identifying whether the child common respiratory tract infection pathogen or nucleic acid product thereof is contained in the sample to be tested;

[0039] D4) preparing to identify or assist in identifying whether the child common respiratory tract infection pathogen or nucleic acid product thereof is contained in the sample to be tested.

[0040] In the above, the child common respiratory tract pathogen is at least one of the following 21 child common respiratory tract pathogens: SPN, HIN, PAE, SA, KPN, ECO, ABA, MC, ECL, SPY, SMA, RSVA, ADVE, IFA, IFB, PIV1, PIV2, PIV3, ADVC, RSVB and ADVB.

[0041] The present application develops a 23-fold child common respiratory tract pathogen nucleic acid mass spectrometry detection method (CCRP-MS), which can help doctors to quickly make etiological diagnosis, and can simultaneously detect 21 child common respiratory tract pathogens, including 11 bacteria and 10 viruses. The detection limit of CCRP-MS is as low as 1 copy / ul, and there is no cross reaction with other pathogens. At the same time, CCRP-MS and real-time fluorescent quantitative reverse transcriptase polymerase chain reaction (RT-PCR) are used to evaluate the clinical effect of 450 cases of child respiratory samples. The positive coincidence rate of the two methods is 96.0%, and the multiple pathogen infection rate is 7.1%. CCRP-MS is a rapid, sensitive, accurate and inexpensive method for detecting child common respiratory tract pathogens, which can provide a new detection method for laboratory diagnosis of respiratory tract infection. The main innovations of the present application are as follows: simultaneous detection of common bacteria and viruses; MRT-PCR and MALDI-TOF MS technology are combined to achieve the purpose of simultaneous detection of common pathogens in child respiratory specimens.

[0042] In addition, the method can realize multiple detection of pathogens. The new method CCRP-MS has no cross reaction in and out of the system, and its sensitivity can be as low as 1 copy / ul. Therefore, the above experimental results show that the sensitivity and specificity of CCRP-MS method are good, and multiple pathogen infections can be detected at the same time. The main advantage of this method is high throughput, which can automatically analyze 192 samples at a time. In one sample, up to 21 pathogens can be detected simultaneously, which can greatly improve the detection efficiency. The cost of CCRP-MS method is low, and the cost of each pathogen can be as low as 0.2 dollars (excluding nucleic acid extraction cost). Therefore, this method is very advantageous for epidemiological screening and rapid identification of large sample population infection. However, CCRP-MS can only detect and identify known pathogens in the method, so as to achieve qualitative identification of pathogens, but cannot be quantified. Therefore, it requires clinicians to make clinical judgments based on known information.

[0043] In summary, CCRP-MS is a 23-pathogen multiplex assay that combines matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) with multiplex reverse transcription-polymerase chain reaction (MRT-PCR) to directly detect common respiratory pathogens in children. Compared with traditional detection methods, CCRP-MS has the following advantages: fast, sensitive, accurate, inexpensive, and high throughput. This method will help supplement existing detection methods and contribute to the clinical laboratory diagnosis of respiratory infections in children. BRIEF DESCRIPTION OF DRAWINGS

[0044] Figure 1 To determine the lower limit of detection (LOD) of CCRP-MS.

[0045] Figure 2 To determine the intra-system specificity of CCRP-MS.

[0046] Figure 3 To determine the out-of-system specificity of CCRP-MS.

[0047] Figure 4 To determine the repeatability of CCRP-MS.

[0048] Figure 5 To determine the carryover of CCRP-MS. DETAILED DESCRIPTION

[0049] The experimental methods used in the following examples are conventional methods unless otherwise specified.

[0050] The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.

[0051] Statistical analysis in the following examples: SPSS 26.0 software was used to analyze the consistency of the positive rates of samples detected by CCRP-MS and RT-PCR using Kappa test. When the Kappa value is between 0.81 and 1, it is considered that the two methods are almost completely consistent.

[0052] Ethics in the following examples: the ethics approval came from the Ethics Committee of Beijing Children's Hospital, Capital Medical University. In this study, all the samples used were residual samples after clinical detection, so the informed consent was waived.

[0053] Example 1, establishment of CCRP-MS method and design of primers

[0054] CCRP-MS is a pathogen multiplex assay that combines matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) with multiplex reverse transcription-polymerase chain reaction (MRT-PCR).

[0055] I. Design and synthesis of CCRP-MS primers

[0056] Target gene sequences of pathogens were downloaded from NCBI (https: / / www.ncbi.nlm.nih.gov / ), at least 300 sequences for each pathogen to ensure their universal representation. Multiple sequence alignment was performed using MAFFT, and highly homologous sequences were selected as candidate regions for primer design (https: / / www.ebi.ac.uk / Tools / msa / mafft / ). Primers and single base extension primers for multiplex analysis were designed using MassARRAY Assay Design 3.1 software (Table 1). Gapdh was used as an internal control gene, and BLAST was used to check the specificity of primers and probes (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi). All primers were synthesized by Beijing Huada High-tech Co., Ltd.

[0057] Table 1 Primer sequences for nucleic acid mass spectrometry

[0058]

[0059]

[0060]

[0061] In the above table, the forward primers from top to bottom are sequence 1-sequence 23, the reverse primers from top to bottom are sequence 24-sequence 46, and the single base extension primers from top to bottom are sequence 47-sequence 69.

[0062] a HIN, Haemophilus influenzae; PAE, Pseudomonas aeruginosa; SA, Staphylococcus aureus; KPN, Klebsiella pneumoniae; ECO, Escherichia coli; ABA, Acinetobacter baumannii; MC, Moraxella catarrhalis; SPY, Streptococcus pyogenes; SMA, Stenotrophomonas maltophilia; RSVA, Respiratory syncytial virus A; ADVE, Adenovirus E; IFA, Influenza A; IFB, Influenza B; PIV1, Parainfluenza virus 1; PIV3, Parainfluenza virus 3; ADVC, Adenovirus C; GAPDH2, Glyceraldehyde-3-phosphate dehydrogenase 2; ADVB, Adenovirus B; ECL, Enterobacter cloacae; SPN, Streptococcus pneumoniae; PIV2, Parainfluenza virus 2; RSVB, Respiratory syncytial virus B; GAPDH1, Glyceraldehyde-3-phosphate dehydrogenase 1.

[0063] Verification RT-PCR study design: Target gene sequence download, alignment and specificity check were performed using the same method as described above. Primer design was performed using SnapGene and Oligo 7 software, and all primers were synthesized by Beijing Huada Company Limited.

[0064] Table 2 Verification RT-PCR primers

[0065]

[0066]

[0067] a SPN, Streptococcus pneumoniae; HIN, Haemophilus influenzae; PAE, Pseudomonas aeruginosa; SA, Staphylococcus aureus; KPN, Klebsiella pneumoniae, ECO, Escherichia coli; ABA, Acinetobacter baumannii; MC, Moraxella catarrhalis; ECL, Enterobacter cloacae; SPY, Streptococcus pyogenes; SMA, Stenotrophomonas maltophilia; RSV, Respiratory syncytial virus; ADV, Adenovirus; IFA, Influenza A virus; IFB, Influenza B virus; PIV1, Parainfluenza virus type 1; PIV2, Parainfluenza virus type 2; PIV3, Parainfluenza virus type 3.

[0068] II. Establishment of CCRP-MS method

[0069] The CCRP-MS method is a combination of matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry system and multiplex reverse transcription polymerase chain reaction (MRT-PCR) to simultaneously detect 21 common respiratory tract bacteria and viruses in children.

[0070] The 21 common respiratory tract bacteria and viruses in children include 11 bacteria (Streptococcus pneumoniae (SPN), Haemophilus influenzae (HIN), Pseudomonas aeruginosa (PAE), Staphylococcus aureus (SA), Klebsiella pneumoniae (KPN), Escherichia coli (ECO), Acinetobacter baumannii (ABA), Moraxella catarrhalis (MC), Enterobacter cloacae (ECL), Streptococcus pyogenes (SPY), and Stenotrophomonas maltophilia (SMA)) and 10 viruses (Respiratory syncytial virus A (RSVA), Respiratory syncytial virus B (RSVB), Adenovirus B (ADVB), Adenovirus C (ADVC), Adenovirus E (ADVE), Influenza A virus (IFA), Influenza B virus (IFB), Parainfluenza virus type 1 (PIV1), Parainfluenza virus type 2 (PIV2), and Parainfluenza virus type 3 (PIV3)).

[0071] Considering the large number of virus subtypes, 21 pathogens were divided into two groups, namely well1 16 kinds and well2 5 kinds of pathogens. Among them, well1 includes: SMA, ABA, PIV1, SPY, ECO, PAE, MC, RSVA, IFA, ADVE, HIN, SA, ADVC, IFB, KPN, PIV3; well2 includes: ADVB, ECL, SPN, PIV2, RSVB.

[0072] And Gapdh was used as an internal quality control target gene, and the corresponding primers GAPDH1 and GAPDH2 are shown in Table 1.

[0073] 1. Plasmid synthesis and dilution

[0074] The relatively conserved region sequences in the target genes of different pathogens were selected and constructed into pUC57 vectors to obtain different pathogen plasmids, which were synthesized by Beijing Lihe Huada Co., Ltd. The following Table 3 shows the selection of relatively conserved region sequences in the target genes of 21 kinds of pathogens.

[0075] Table 3 is the conserved region sequence for constructing plasmid

[0076]

[0077]

[0078]

[0079]

[0080] The different pathogen plasmids were sequentially diluted by 10 times in enzyme-free and nucleic acid-free water, and the plasmid concentration was quantified using Qubit.

[0081] 2. CCRP-MS method

[0082] 1) MRT-PCR reaction

[0083] Using the above pathogen plasmids or nucleic acids of the samples to be tested as templates, the forward primers and reverse primers corresponding to the well1 pathogenic bacteria shown in Table 1 and the forward primers and reverse primers corresponding to the well2 pathogenic bacteria were used for 2-group MRT-PCR amplification, to obtain the MRT-PCR reaction products of well1 and the MRT-PCR reaction products of well2. Enzyme-free and nucleic acid-free water was used as a negative control.

[0084] The above sample to be tested can be a pure culture of pathogen, oropharyngeal swab, nasopharyngeal swab, tracheal secretion, bronchial secretion or sputum of infected or suspected infected pathogen;

[0085] The above pathogen plasmid is a diluted plasmid.

[0086] The MRT-PCR reaction system: 2 μl template, 1.5 μl nucleic acid-free water, 0.5 μl RT-RCR enzyme, 5 μl buffer (all reagents from the Time-of-Flight Mass Spectrometry Nucleic Acid Detection Universal Kit (RNA), Beijing Huada Gibaibio Biotechnology Co., Ltd. Product Catalog No. 0612054) and 1 μl primer mixture (the final concentration of each primer in the reaction system is 500 nmol) were mixed together.

[0087] The primer mixture in the MRT-PCR system corresponding to Well1 was mixed with the same volume of the forward and reverse primers corresponding to the 17 pathogens in Table 1 in the well1 group, dissolved in water, and the resulting mixture was obtained.

[0088] The primer mixture in the MRT-PCR system corresponding to Well2 was mixed with the same volume of the forward and reverse primers corresponding to the 6 pathogens in Table 1 in the well2 group, dissolved in water, and the resulting mixture was obtained.

[0089] The MRT-PCR reaction conditions were: 50°C for 30 minutes, 94°C for 10 minutes, 45 cycles (94°C for 30 seconds, 56.5°C for 30 seconds and 72°C for 30 seconds), and 72°C for 7 minutes.

[0090] 2) Shrimp alkaline phosphatase (SAP) digestion

[0091] 3.4 μl of nucleic acid reaction solution 2 and 0.6 μl of nucleic acid reaction solution 3 (all reagents from the Time-of-Flight Mass Spectrometry Nucleic Acid Detection Universal Kit (RNA), Beijing Huada Gibaibio Biotechnology Co., Ltd. Product Catalog No. 0612054) were added to the MRT-PCR reaction products of well1 and well2, respectively, to carry out the digestion reaction, and the digestion products of well1 and well2 were obtained.

[0092] The conditions for the above digestion reaction were as follows: 37°C for 40 minutes, 85°C for 5 minutes. The above conditions were amplified to remove unbound primers and dNTPs.

[0093] 3) Single base extension (SBE) reaction

[0094] To the digestion product of well 1 and the digestion product of well 2 in the above step, respectively, add 0.8 μl of nucleic acid reaction solution 4, 0.4 μl of nucleic acid reaction solution 5, 0.9 μl of nucleic acid reaction solution 6 (all reagents are from Time-of-Flight Mass Spectrometry Nucleic Acid Detection Universal Kit (RNA), Beijing Huada Gibibo Biotechnology Co., Ltd. Product Catalog No. 0612054) and 1.9 μl of corresponding SBE primer mixture, to obtain a single base extension (SBE) reaction system; perform amplification according to the following single base extension (SBE) reaction procedure to obtain a single base extension product of well 1 and a single base extension product of well 2.

[0095] The SBE primer mixture in the single base extension (SBE) reaction system of well 1 is to mix the single base extension primers corresponding to the 17 pathogens in well 1 group in Table 1 in equal volumes after dissolving in water, and the SBE primer preparation is shown in Table 4.

[0096] The SBE primer mixture in the single base extension (SBE) reaction system of well 2 is to mix the single base extension primers corresponding to the 6 pathogens in well 2 group in Table 1 in equal volumes after dissolving in water, and the SBE primer preparation is shown in Table 4.

[0097] Table 4 is the SBE primer configuration table

[0098]

[0099]

[0100] The above single base extension (SBE) reaction procedure is as follows: 95°C for 30 seconds, 40 cycles (94°C for 5 seconds, 52°C for 5 seconds and 80°C for 5 seconds), and 72°C for 3 minutes.

[0101] The single base extension product of well 1 and the single base extension product of well 2 obtained above are respectively desalted and purified with resin to obtain a purified product of well 1 and a purified product of well 2.

[0102] The specific method of the above resin desalting purification is as follows: add 32 μl of enzyme-free nucleic acid-free water to the single base extension reaction product in the above step, and then add 20 μl of resin (all reagents are from Time-of-Flight Mass Spectrometry Nucleic Acid Detection Universal Kit (RNA), Beijing Huada Gibibo Biotechnology Co., Ltd. Product Catalog No. 0612054), place the sample plate in a horizontal inversion shaker, rotate it along the long axis at a speed of 15 rpm at room temperature for 40 min, and centrifuge it at a speed of 4000 rpm for 5 min. The supernatant is the purified product.

[0103] 4) Nucleic acid mass spectrometry detection

[0104] The purified product of well 1 and the purified product of well 2 obtained in the above 3) were spotted to the target plate chip using a full-automatic sample processing system (MSP96) for detection in a mass spectrometer (GBI MS ToF-1000). The negative control was a purified product obtained according to the steps of 1) to 3) above using no enzyme and no nucleic acid water as the template.

[0105] Data collection and analysis were performed using the Nutyper software of the nucleic acid mass spectrometer.

[0106] The results corresponding to the purified product of well 1 and the purified product of well 2 were detected. If the detection result of the sample to be tested met the following standard: compared with the negative control, the single-base extension primer peak in the purified product disappeared or decreased, and the product peak appeared in the purified product, then the sample to be tested contained or was suspected to contain the target pathogen corresponding to the product peak, i.e., it was positive. If the detection result of the sample to be tested did not meet the above standard, then the sample to be tested did not contain or was suspected not to contain the target pathogen corresponding to the product peak, i.e., it was negative.

[0107] Example 2, Application of the CCRP-MS method

[0108] I. Sensitivity evaluation of the CCRP-MS method

[0109] Different pathogenic bacterial plasmids were diluted by 10-fold gradient (1-10 4 copies / μl) using no enzyme and no nucleic acid water to evaluate the sensitivity thereof.

[0110] The steps of 2 in 2 of Example 1 were adopted, and the above diluted plasmids were used as templates for CCRP-MS detection.

[0111] The results are shown in Table 5 and Figure 1well2 primer amplification result (welll negative) of SPN, Streptococcus pneumoniae; (B) welll primer amplification result (well2 negative) of HIN, Haemophilus influenzae; (C) welll primer amplification result (well2 negative) of PAE, Pseudomonas aeruginosa; (D) welll primer amplification result (well2 negative) of SA, Staphylococcus aureus; (E) welll primer amplification result (well2 negative) of KPN, Klebsiella pneumoniae; (F) welll primer amplification result (well2 negative) of ECO, Escherichia coli; (G) welll primer amplification result (well2 negative) of ABA, Acinetobacter baumannii; (H) welll primer amplification result (well2 negative) of MC, Moraxella catarrhalis; (I) well2 primer amplification result (welll negative) of ECL, Enterobacter cloacae; (J) welll primer amplification result (well2 negative) of SPY, Streptococcus pyogenes; (K) welll primer amplification result (well2 negative) of SMA, Stenotrophomonas maltophilia; (L) welll result (well2 negative) of RSV A, Respiratory Syncytial Virus A; (M) welll primer amplification result (well2 negative) of ADVE, Adenovirus E; (N) welll primer amplification result (well2 negative) of IFA, Influenza A; (O) welll primer amplification result (well2 negative) of IFB, Influenza B; (P) welll primer amplification result (well2 negative) of PIVl, Parainfluenza Virus 1; (Q) well2 primer amplification result (welll negative) of PIV2, Parainfluenza Virus 2; (R) welll primer amplification result (well2 negative) of PIV3, Parainfluenza Virus 3; (S) welll primer amplification result (well2 negative) of ADVC, Adenovirus C; (T) well2 primer amplification result (welll negative) of RSVB, Respiratory Syncytial Virus B; (U) well2 primer amplification result (welll negative) of ADVB, Adenovirus B; white arrow indicates disappearance or reduction of single base extension primer peak, black arrow indicates appearance of product peak; it can be seen that the detection lower limit (LOD) is 1 to 10 3 copies / μl. The well figures of the negative results of the above-mentioned various pathogens are not shown.

[0112] Table 5 is the detection lower limit (LOD) of CCRP-MS

[0113]

[0114] a SPN, Streptococcus pneumoniae; HIN, Haemophilus influenzae; PAE, Pseudomonas aeruginosa; SA, Staphylococcus aureus; KPN, Klebsiella pneumoniae; ECO, Escherichia coli; ABA, Acinetobacter baumannii; MC, Moraxella catarrhalis; ECL, Enterobacter cloacae; SPY, Streptococcus pyogenes; SMA, Stenotrophomonas maltophilia; RSVA, Respiratory syncytial virus type A; ADVE, Adenovirus type E; IFA, Influenza A virus; IFB, Influenza B virus; PIV1, Parainfluenza virus type 1; PIV2, Parainfluenza virus type 2; PIV3, Parainfluenza virus type 3; ADVC, Adenovirus type C; RSVB, Respiratory syncytial virus type B; ADVB, Adenovirus type B.

[0115] II. Specificity

[0116] The assessment is divided into in-system and out-of-system specificity evaluation:

[0117] 1. System specificity

[0118] Five concentrations of 10 were mixed in equal volumes. 4 Two sets of intrasystem plasmids were obtained by taking copies / μl of the two sets of system plasmids, resulting in two mixtures.

[0119] The plasmids within the system are divided into the following two groups: Group 1 contains pathogen plasmids of Stenotrophomonas maltophilia SMA, Acinetobacter baumannii ABA, Pseudomonas aeruginosa PAE, Staphylococcus aureus SA, and Klebsiella pneumoniae KPN; Group 2 contains pathogen plasmids of adenovirus type B ADVB, Enterobacter cloacae ECL, Streptococcus pneumoniae SPN, Parainfluenza virus type 2 PIV2, and Respiratory syncytial virus type B RSVB.

[0120] Using the two mixtures mentioned above as templates, CCRP-MS detection was performed using step 2 in Example 1, and the samples were spotted for detection.

[0121] The results are as follows Figure 2 As shown, Figure 2 A indicates that the results of well 1 testing for group 1 (well 2 test result was negative) include (A) SMA, Stenotrophomonas maltophilia; (B) ABA, Acinetobacter baumannii; (C) PAE, Pseudomonas aeruginosa; (D) SA, Staphylococcus aureus; and (E) KPN, Klebsiella pneumoniae. Figure 2B represents (F) ADVB, Adenovirus B; (G) ECL, Enterobacter cloacae; (H) SPN, Streptococcus pneumoniae; (I) PIV2, Parainfluenza virus type 2; (J) RSVB, Respiratory syncytial virus type B, well2 detection results of group 2 (welll detection results are negative), white arrow indicates the disappearance or reduction of single base extension primer peak, black arrow indicates the appearance of product peak; all results are positive and no other pathogen cross reaction. The well figure of each pathogen negative result is not shown.

[0122] 2. System specificity

[0123] Mix 10 concentrations of 10 copies / μl of nucleic acid of other pathogens outside the system or diluted plasmid in equal volume to obtain a mixture; 4 copies / μl of nucleic acid of other pathogens outside the system or diluted plasmid in equal volume to obtain a mixture;

[0124] The above mixture is a nucleic acid mixture of hepatitis B virus, coxsackie virus, dengue virus, hantavirus, mycobacterium tuberculosis, human papilloma virus, neisseria gonorrhoeae, staphylococcus epidermidis, enterococcus faecalis and candida albicans.

[0125] Using the mixture as a template, the steps in 2 of 2 in Example 1 are used to perform CCRP-MS detection, spot detection.

[0126] The results are shown in Figure 3 A indicates the welll detection results of the mixture, B indicates the well2 detection results of the mixture, white arrow indicates the disappearance or reduction of single base extension primer peak, and the results are all negative; no product peak appears, the single base extension primer peak does not disappear or reduce, and the results are all negative.

[0127] Three, repeatability

[0128] The repeatability of the minimum detection limit test method of the two intrasystem plasmids is used.

[0129] 1 copy / μl of influenza A virus plasmid and 1 copy / μl of streptococcus pneumoniae plasmid are used as templates, and the steps in 2 of 2 in Example 1 are used to perform CCRP-MS detection, spot detection:

[0130] The results are shown in Figure 4 A indicates the welll detection results of the mixture, B indicates the well2 detection results of the mixture, white arrow indicates the disappearance or reduction of single base extension primer peak, and the results are all negative; no product peak appears, the single base extension primer peak does not disappear or reduce, and the results are all negative.

[0131] The result of well 2 of IFA (Influenza A virus) with a concentration of 1 copy / μl was negative, and the result of well 1 of SPN (Streptococcus pneumoniae) with a concentration of 1 copy / μl was negative.

[0132] III. Carryover detection

[0133] Ten 5 copies / μl of Staphylococcus aureus plasmid and 10 5 copies / μl of Respiratory Syncytial Virus B plasmid were used as templates, and the CCRP-MS detection was performed using the procedure of Example 1, Step 2, respectively. After 3 groups of sample points were arranged and detected, respectively, and negative controls (nuclease-free water) were repeated, whether the system had carryover was detected.

[0134] The results are shown in Figure 5 A indicates the result of well 1 (well 2 was negative) with a template of 10 5 copies / μl of SA (Staphylococcus aureus), B indicates the result of well 1 (well 2 was negative) with a template of nuclease-free water, C indicates the result of well 2 (well 1 was negative) with a template of 10 5 copies / μl of RSVB (Respiratory Syncytial Virus B), and D indicates the result of well 2 (well 1 was negative) with a template of nuclease-free water. The white arrow indicates that the single-base extension primer peak disappears or decreases, and the black arrow indicates that the product peak appears, showing that the method does not have carryover problems.

[0135] Example 3, Clinical sample application of CCRP-MS method

[0136] I. Nucleic acid acquisition of clinical samples

[0137] 1. Acquisition of clinical samples

[0138] Four hundred and fifty samples were collected from children (15 days-16 years old) with respiratory symptoms who visited the Beijing Children's Hospital, Capital Medical University from 2020 to 2021 (ethical approval from the Ethics Committee of Beijing Children's Hospital, Capital Medical University was obtained, and Table 6 shows the number of samples from different pathogens). Sample types included oropharyngeal swabs, nasopharyngeal swabs, tracheal secretions, bronchial secretions, and sputum.

[0139] Table 6 is the clinical sample used in this study

[0140]

[0141] aSPN, Streptococcus pneumoniae; HIN, Haemophilus influenzae; PAE, Pseudomonas aeruginosa; SA, Staphylococcus aureus; KPN, Klebsiella pneumoniae, ECO, Escherichia coli; ABA, Acinetobacter baumannii; MC, Moraxella catarrhalis; ECL, Enterobacter cloacae; SPY, Streptococcus pyogenes; SMA, Stenotrophomonas maltophilia; RSV, Respiratory syncytial virus; ADV, Adenovirus; IFA, Influenza A virus; IFB, Influenza B virus; PIV1, Parainfluenza virus type 1; PIV2, Parainfluenza virus type 2; PIV3, Parainfluenza virus type 3; Negative control, negative control.

[0142] b In order to show the distribution of pathogens, the multiple infection pathogens are divided into single pathogens for statistics.

[0143] 2. Sample preparation

[0144] Since some samples are very viscous, such as sputum, tracheal secretion, bronchial secretion, etc., a liquefying agent (BASO BC1997) is added to liquefy them into a uniform and thin solution.

[0145] 3. Nucleic acid co-extraction

[0146] EX-48 automatic nucleic acid extraction system (Beijing Huada Gibibo Biotechnology Co., Ltd.) is used to extract 200 microliters of DNA and RNA from each sample for co-extraction, and the nucleic acid of the sample to be tested is obtained.

[0147] II. CCRP-MS detection

[0148] The nucleic acid of the sample to be tested is used as a template, and the steps of 2 in II of Example 1 are used to detect CCRP-MS.

[0149] At the same time, the nucleic acid of the sample to be tested is detected by RT-PCR method.

[0150] The experimental results are shown in Table 7 (negative results are not shown). The positive rate of CCRP-MS and RT-PCR detection of multiple infections is 7.1%, and the details are shown in Table 8. The positive coincidence rate of 21 pathogens detected by the two methods is 96% (432), and the inconsistent rate is 4% (18). The inconsistent results are reconfirmed by sequencing method, and Table 9 provides detailed information of the inconsistent results during verification. The positive rate of CCRP-MS method is 84.7%, and the positive rate of RT-PCR method is 83.3%. There is significant consistency between the two methods (Kappa = 0.851, SE of kappa = 0.034, 95% confidence interval is 0.784-0.918), and the result comparison and analysis of the two methods are shown in Table 10.

[0151] Table 7. CCRP-MS and RT-PCR parallel detection of clinical samples

[0152]

[0153]

[0154] Table 8. Confirmation of CCRP-MS and RT-PCR discordant results

[0155] Sample CCRP-MS results RT-PCR results Sequencing results 1 Streptococcus pneumoniae - Streptococcus pneumoniae 2 Enterobacter cloacae - Enterobacter cloacae 3 Streptococcus pneumoniae - Streptococcus pneumoniae 4 Streptococcus pneumoniae - Streptococcus pneumoniae 5 Streptococcus pneumoniae - Streptococcus pneumoniae 6 Streptococcus pneumoniae - Streptococcus pneumoniae 7 Enterobacter cloacae - Enterobacter cloacae 8 Streptococcus pneumoniae - Streptococcus pneumoniae 9 - Haemophilus influenzae Haemophilus influenzae 10 - Influenza A virus Influenza A virus 11 - Influenza A virus Influenza A virus 12 - Parainfluenza virus 1 Parainfluenza virus 1 13 Parainfluenza virus 1 - Parainfluenza virus 1 14 Parainfluenza virus 3 - Parainfluenza virus 3 15 - Parainfluenza virus 3 Parainfluenza virus 3 16 - Adenovirus Adenovirus 17 Respiratory syncytial virus - Respiratory syncytial virus 18 Respiratory syncytial virus - Respiratory syncytial virus

[0156] Table 9. Multiple infections with concordant CCRP-MS and RT-PCR detection results a

[0157]

[0158]

[0159] a The total number of samples was 32.

[0160] Table 10. Summary table of CCRP-MS and RT-PCR results of clinical samples

[0161]

[0162] Kappa = 0.851, SE of kappa = 0.034, 95% confidence interval 0.784-0.918, there is significant concordance between the two methods.

[0163] The results above show that all CCRP-MS detection results have been confirmed by the RT-PCR method.

Claims

1. A primer set, comprising a specific primer set and a single-base extension primer set; The specific primer set consists of single-stranded DNA molecules shown in sequences 1-16, sequences 24-39, sequences 18-22, and sequences 41-45. The single-base extension primer set consists of single-stranded DNA molecules shown in sequences 47-62 and 64-68.

2. MRT-PCR reagent, containing the specific primer set as described in claim 1 and the single-base extension primer set as described in claim 1.

3. The use of the primer set of claim 1 or the MRT-PCR reagent of claim 2 in any of the following: C1) Prepare reagent kits for detecting or assisting in the detection of common respiratory pathogens in children or their nucleic acid samples; C2) Prepare screening or auxiliary screening kits for common respiratory infection pathogens in children or their nucleic acid reagents; C3) Prepare a kit for identifying or assisting in the identification of whether the sample to be tested contains common respiratory pathogens in children or their nucleic acid; The common respiratory pathogens in children are at least one of the following 21 common respiratory pathogens in children: SPN, HIN, PAE, SA, KPN, ECO, ABA, MC, ECL, SPY, SMA, RSVA, ADVE, IFA, IFB, PIV1, PIV2, PIV3, ADVC, RSVB, and ADVB.

4. A reagent kit comprising the following components: B1) The primer set as described in claim 1; Or, B2) the MRT-PCR reagent of claim 2.

5. The reagent kit according to claim 4, characterized in that: The kit also includes the following: B3) Shrimp alkaline phosphatase; B4) Instruments or reagents required for nucleic acid mass spectrometry detection.

6. The application of the kit according to claim 4 or 5 in any of the following: D1) Preparation of nucleic acid products for the detection or auxiliary detection of common respiratory infection pathogens in children; D2) Prepare screening or auxiliary screening products for common respiratory infection pathogens in children or their nucleic acid products; D3) Preparation for identification or auxiliary identification of whether the sample to be tested contains common respiratory pathogens in children or their nucleic acid products; The common respiratory pathogens in children are at least one of the following 21 common respiratory pathogens in children: SPN, HIN, PAE, SA, KPN, ECO, ABA, MC, ECL, SPY, SMA, RSVA, ADVE, IFA, IFB, PIV1, PIV2, PIV3, ADVC, RSVB, and ADVB.

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