CN114350857A - Primer combination, kit and detection method for mass spectrometry detection of multiple respiratory viruses - Google Patents
Primer combination, kit and detection method for mass spectrometry detection of multiple respiratory viruses Download PDFInfo
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Abstract
The invention discloses a primer combination, a kit and a detection method for mass spectrometric detection of various respiratory viruses, and relates to the technical field of molecular biology detection. The kit comprises 48 amplification primers and 24 mass probe extension primers, and can realize typing identification of various respiratory viruses by combining a multiplex PCR technology and a MALDI-TOF technology. The detection flux is high, and the method is more suitable for large-scale pathogen screening.
Description
Technical Field
The invention relates to the technical field of molecular biology detection. More particularly, relates to a primer combination, a kit and a detection method for mass spectrometry detection of various respiratory viruses.
Background
The types of pathogens that cause respiratory infections in humans are diverse, viruses are an important causative agent, and about 80% of respiratory infections are caused by respiratory viruses. The series of viruses is very diverse, the most common of which typically includes: influenza viruses (influenzaviruses, IFVs) of the family Orthomyxoviridae (Orthomyxoviridae), Respiratory Syncytial Viruses (RSV) of the family Paramyxoviridae (Paramyxoviridae), parainfluenza viruses (parainfluenza viruses, PIVs), Human Metapneumoviruses (HMPV), human rhinoviruses (hramnaviruses, HRVs) of the family Picornaviridae (picornaviruses), human coronaviruses (human coronaviruses, HCoVs) of the family Picornaviridae (adenoviruses, adnoviruses) of the family adenoviruses (adenoviruses), and human bocaviruses (hbovaviruses) of the family subviraceae (paraviridae). Although respiratory viruses are diverse in kind, their infection is similarly characterized. First, respiratory viruses are highly contagious and are very easily transmitted in places where people gather, such as schools and hospitals. Secondly, symptoms caused by respiratory virus infection are not specific, and are mostly presented as symptoms of upper respiratory infection, such as common cold, sphagitis and the like. Third, the population is generally susceptible to respiratory pathogens, the rate of viral variation is rapid, the immunity achieved by vaccines is not long-lasting, and respiratory infections are likely to recur. 2019 the novel coronavirus is highly contagious and has a high transmission speed, especially for people with low immunity, such as the elderly, immunosuppression and basic patients are easy to form secondary infection sometimes, cause serious damage to the lung and other organs, develop severe damage and even endanger life.
The current main method for detecting nucleic acid of respiratory viruses is a fluorescent quantitative RT-PCR method. However, due to the limitation of the fluorescent channel, only 1-2 viruses can be detected at one time, and the detection flux of each sample is low, so that the clinical rapid and accurate large-scale case screening is not facilitated; NGS high-throughput sequencing is also an important technology for detecting viruses, and the accurate identification of the viruses and subtypes can be realized through full sequencing, but the experimental operation is complex, the overall process time is long, and the sample detection cost is increased. Therefore, for the detection of respiratory pathogens, the detection weight and the detection flux must be considered together.
Disclosure of Invention
The invention aims to provide a primer combination for mass spectrometric detection of multiple respiratory viruses and a detection kit containing the primer combination, which improve the screening and diagnosis capability of the respiratory viruses.
The invention also aims to provide a method for carrying out mass spectrometry detection on various respiratory virus nucleic acids by using the kit. The method has the advantages of strong specificity and high sensitivity of respiratory virus detection.
In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect, the invention provides a primer combination for mass spectrometric detection of multiple respiratory viruses, comprising 48 amplification primers and 24 mass probe extension primers, wherein the nucleotide sequences of the amplification primers are shown as SEQ ID Nos. 1-48, and are specifically shown in Table 1; the nucleotide sequence of the mass probe extension primer is shown in SEQ ID Nos. 49-72, and is specifically shown in Table 2.
TABLE 1 amplification primer sequences
TABLE 2 Mass Probe Extension (MPE) primer sequences
The invention combines the multiplex PCR technology with MALDI-TOF mass spectrum technology, i.e. PCR-micro sequencing is adopted to carry out the typing detection of various respiratory pathogens. In the invention, a series of PCR amplification primers and quality probe extension (MPE) primers for detecting 20 respiratory viruses are designed by performing sequence analysis on 20 respiratory viruses, selecting and detecting a target gene and a specific mutation site, and internal reference genes such as RNaseP and HBB are also arranged to monitor whether sampling and amplification experiments of samples are successful, so that the detection sensitivity is greatly improved, high-throughput detection in a short time can be realized, and the method is more suitable for large-scale detection and screening of suspected virus cases.
The invention also provides application of the primer combination in preparation of a plurality of respiratory virus nucleic acid mass spectrum detection products.
According to a specific embodiment of the invention, the invention provides a kit for mass spectrometric detection of multiple respiratory virus nucleic acids, which comprises the primer combination.
Furthermore, the kit also comprises an RT-PCR reaction reagent, a dephosphorylation reaction reagent and a mass probe extension reaction reagent.
In a second aspect, the present invention provides a method for detecting multiple respiratory viruses, the method using the above kit, specifically comprising:
(1) carrying out RT-PCR amplification reaction on a sample to be detected by using 48 amplification primers;
(2) dephosphorizing the amplification product obtained in the step (1) by using alkaline phosphatase;
(3) performing single base extension on the dephosphorylated product obtained in the step (2) by using 24 mass probe extension primers;
(4) performing resin desalination and purification on the extension product obtained in the step (3);
(5) detecting by mass spectrum, and determining the pathogen type.
Further, the plurality of respiratory viruses are respiratory syncytial viruses a and B (RSV-a and RSV-B), parainfluenza virus types 1 to 4 (PIV1, PIV2, PIV3, PIV4), human metapneumoviruses a and B (HMPV-a and HMPV-B), human bocavirus types 1 and 2 (HBoV1 and HBoV2), rhinovirus (HRV), influenza virus universal (FluA), influenza virus H1N1(FluA-H1N1), influenza virus H3N2(FluA-H3N2), influenza virus H1N12009 (FluA-H1N 12009), influenza virus (FluB), coronavirus-SARS, coronavirus-MERS, novel coronavirus SARS-CoV-2, and Adenovirus (ADV).
Further, the mass spectrometry detection adopts matrix-assisted laser desorption ionization time-of-flight mass spectrometry MALDI-TOF MS.
Further, the alkaline phosphatase is shrimp alkaline phosphatase.
The invention has the following beneficial effects:
the invention can realize the detection of various respiratory viruses at one time by combining the multiplex PCR technology and the MALDI-TOF technology. For common viruses infected by respiratory tract, the PCR-MALDI micro-sequencing technology adopted by the invention uses specific primer combination, has high detection flux, can detect 20 viruses at one time, can obviously reduce false negative results, and particularly greatly reduces the detection cost of each site on average; on the other hand, the invention has less requirements on samples and high sensitivity, the lower limit of sample detection reaches 10-100 copies/mu L, and the invention is more suitable for large-scale pathogen screening.
Drawings
The following describes embodiments of the present invention in further detail with reference to the accompanying drawings.
FIG. 1 shows the results of primer specificity verification.
Figure 2 shows the precision experiment 24 heavy particle amplification mass spectrum.
Figure 3 shows an ADV positive amplification mass spectrum.
FIG. 4 shows FluA site-positive amplification mass spectra.
FIG. 5 shows the FluA-H3N2 site positive amplification mass spectrum.
FIG. 6 shows a FluB site positive amplification mass spectrum.
FIG. 7 shows FluA site-positive amplification mass spectra.
FIG. 8 shows a spectrum of FluA-H1N12009 site positive amplification.
FIG. 9 shows RSV-B site positive amplification mass spectra.
Detailed Description
In order to more clearly illustrate the invention, the invention is further described below with reference to preferred embodiments and the accompanying drawings. Similar parts in the figures are denoted by the same reference numerals. It is to be understood by persons skilled in the art that the following detailed description is illustrative and not restrictive, and is not to be taken as limiting the scope of the invention.
Reagents and instruments used in the experimental procedure:
viral RNA Mini Kit (52904): qiagen Enterprise management (Shanghai) Co., Ltd;
nucleic acid detection kit (pathogenic microorganism) (QT-SJ 12-RTs): science and technology (Qingdao) Inc.; the kit comprises the following components:
a1 component: RT-PCR enzyme, a2 component: RT-PCR buffer solution;
a3 component: SAP enzyme, a4 component: an SAP buffer;
a5 component: MPE enzyme, a6 component: MPE buffer, a7 component: e _ ddNTPmix;
a8 component: a matrix liquid;
plasmid and primer synthesis: biometrics (Shanghai) Inc.;
VeritiTM96-well thermal cycler: sammer Feishel technologies, Inc.;
NanoDropTMone spectrophotometer: sammer Feishel technologies, Inc.;
QuanTOF I mass spectrometer: science and technology (Qingdao) Inc.
Example 1 amplification primer and Mass Probe extension primer design
The complete sequences of 20 pathogen strains are downloaded from NCBI, 5-10 strains are downloaded for each strain, the BioEdit sequence Alignment Editor software is used for sequence Alignment, key genes such as N of SARS-CoV-2, ORF1ab and the like are mainly compared, target genes with intra-species conservation and inter-species specificity are selected for detection (see Table 3), and human RNaseP and HBB are selected as internal references.
Multiplex PCR Primer design was performed using Primer3 on-line web pages. Specific amplification primers are shown in Table 1 above. Mutation sites are selected and detected, and genetic locus typing system software (intelligent melt biotechnology (Qingdao) Co., Ltd.) is adopted to design quality probe extension (MPE) primers, which is shown in the table 2. The designed PCR primers and MPE primers were synthesized by engineering bioengineering (Shanghai) Inc. for plasmid and primer synthesis, respectively.
TABLE 3 target genes for pathogen-corresponding detection
Example 2 detection method establishment
1. And (5) diluting the plasmid.
The plasmid dry powder was diluted to 100ng/ul with water and accurately quantified using Nanodrop. The copy number contained in the plasmid was calculated from the plasmid sequence. Various concentrations, 10 each, were used in the experiments5copies/μL，104copies/μL，103copies/μL，102copies/μL，101copies/μL，100copy/μL。
2. Primer dilution and mixing.
Preparing PCR primer mixed solution: after the PCR primer sequences of example 1 were synthesized, the dry powder was dissolved in 100. mu.M stock solution using water. The stock solution is taken out and mixed to prepare a primer mixture solution with the final concentration of the primer of each site ranging from 0.5 mu M to 5 mu M.
Preparing MPE primer mixed solution: after the mass probe extension primer sequences of example 1 were synthesized, the dry powder was dissolved in 500. mu.M stock solution using water. Taking out the primer stock solution, mixing, and preparing a primer mixture solution with the final concentration of the primer of each site ranging from 5 mu M to 15 mu M.
RT-PCR reaction
(1) An RT-PCR system was configured using a nucleic acid detection kit (pathogenic microorganism) (QT-SJ12-RTs), as shown in Table 4.
TABLE 4 RT-PCR SYSTEM CONFIGURATION TABLE
| Reagent composition | Volume (μ L) |
| RT-PCR enzymes | 1 |
| RT-PCR buffer | 12.5 |
| PCR primer mixture | 5 |
| Form panel | 6.5 |
| Total | 25 |
(2) The RT-PCR program was run as per Table 5.
TABLE 5 RT-PCR temperature-controlled reaction procedure
(3) After the reaction was completed, 5. mu.L of the amplified product was taken out for the subsequent experimental reaction.
4. Shrimp Alkaline Phosphatase (SAP) dephosphorylation treatment
(1) A nucleic acid detection kit (pathogenic microorganism) (QT-SJ12-RTs) is utilized to configure an SAP reaction system. The SAP reaction was prepared as in Table 6.
TABLE 6 SAP SYSTEM CONFIGURATION TABLE
TABLE 6 SAP SYSTEM CONFIGURATION TABLE
(2) mu.L of SAP reaction solution was added to 5. mu.L of the amplification product taken out in the previous step, and placed on a PCR instrument to run SAP dephosphorylation reaction.
TABLE 7 SAP temperature control reaction procedure
| Reaction temperature | Time |
| 37℃ | 40min |
| 85℃ | 5min |
| 4℃ | hold |
MPE quality probe extension
(1) And (3) configuring an MPE reaction system by using a nucleic acid detection kit (pathogenic microorganisms) (QT-SJ 12-RTs). The MPE reaction solution was prepared as shown in Table 8.
TABLE 8 MPE SYSTEM CONFIGURATION TABLE
| Reagent composition | Volume (μ L) |
| MPE enzymes | 0.6 |
| MPE buffer solution | 1.4 |
| E_ddNTPmix | 1 |
| MPE primer mixture | 1 |
| Total | 4 |
(2) Add 4. mu.L MPE reaction solution to 7. mu.L dephosphorylated product from the previous step, place on PCR instrument to run MPE reaction.
TABLE 9 MPE temp. CONTROL PROGRAM
6. Resin desalting purification and target plate spotting.
(1) To each reaction well was added 14. mu.L of deionized water.
(2) The resin filled eight tubes were gently inverted and snapped onto the sample plate to ensure that the resin holes were aligned with each hole in the sample. The resin tube was then tapped to drop the resin into the wells of the sample plate.
(3) The sample plate with the resin was placed in an inverted mixer and mixed for 30min at 20 rpm.
(4) After mixing, the sample was centrifuged at 2000rpm for 1min, and 2. mu.L of the supernatant was mixed with an equal volume of the matrix solution.
(5) The mixture was spotted at 1. mu.L onto a target plate.
7. Target plate acquisition and data analysis.
(1) According to the instruction of the QuandTOF 1 instrument, a target plate for co-crystallizing the substrate and the sample is loaded into the instrument, and data acquisition is carried out after the vacuum degree reaches the requirement (BA Gauge is superior to 2e-6 Torr). The acquisition mode of the instrument is a linear positive ion mode, and important parameters are set as follows: accelate Voltage 20kV, Mass Range 3000-11000Da, Laser Frequency 3000Hz, Shots/Spectrum 800, and Laser energy 24 uJ.
(2) After the collection is completed, clicking analysis is performed, the software gives the extension of each position point of each sample, and the detection result of the sample can be checked. The important parameters are set as: SNR: 4.0.
example 3 specificity test
1. The primer combination is amplified without a template to verify the specificity of the primer. The whole experimental flow from RT-PCR reaction to resin desalting purification was run using water instead of sample template as in example 2, which was verified that there was no dimer between multiplex PCR amplification primers and multiplex MPE primers and no non-specific amplification extension products, ensuring the specificity of primer design. The results of the experiment are shown in FIG. 1.
2. The primer specificity was verified using mixed plasmids. One set of PCR design primers and MPE primers for each pathogen theoretically could have an extension result only if the positive plasmid was present. Therefore, single pathogen plasmid is added respectively, the concentration is 10^5 copies/mu L, and the verification result shows that the extension product exists in the corresponding site, and no product is detected in other sites.
3. Other pathogens were used to verify primer specificity. Also capable of causing respiratory tract infections are bacteria (streptococcus pneumoniae, klebsiella pneumoniae, etc.), mycoplasma pneumoniae, chlamydia, etc. The experiment uses pure bacteria cultured mycoplasma pneumoniae to extract nucleic acid, and verifies that no target is detected, so that the false detection result is not reported in a non-specific manner by other pathogenic microorganisms.
Example 4 sensitivity, precision and accuracy experiments
(1) Sensitivity verification
The individual plasmids prepared in example 1 were diluted with water in 10-fold concentration gradients of 10 each5copies/μL，104copies/μL，103copies/μL，102copies/μL，101copies/. mu.L, 100 copy/. mu.L. Six concentrations of plasmid template were amplified and extended, respectively. The vast majority of sites are at 102copies/. mu.L were detected in full and half of the sites were still significantly amplified at 10 copies/. mu.L. Wherein SARS-CoV-2-N is only 105Amplification occurred at copies/. mu.L or more, and the primer concentration was optimized to improve the reaction efficiency of N. The end result is that the lower limit of detection of 20 viruses is between 10-100 copies/. mu.L. See table 10 for details.
TABLE 10 lower detection limits for each pathogen site
(2) Precision verification
The concentration of the mixed plasmid is 100 copies/mu L, 3 times of repeated experiments are carried out, 5 times of repeated experiments are completed in 2 weeks, 24 products can be detected, and the detection result is stable. See fig. 2.
(3) Accuracy verification
3 received extracted nucleic acid samples are identified as positive samples of ADV, FluA and FluB by a fluorescence qPCR method, the method of the embodiment 2 is adopted for detection, the result is that all the positive samples are detected, and the FluA positive sample can be successfully positioned to the H3N2 subtype. The results are shown in FIGS. 3-6.
EXAMPLE 5 sample testing
2 influenza-positive pharyngeal swab samples were obtained from a hospital, subjected to nucleic acid extraction using a Qiagen virus extraction Kit (Viral RNA Mini Kit (52904)), and subjected to respiratory virus nucleic acid mass spectrometry according to the method of example 2.
Results sample No.1 FluA-H1N12009 was positive, as shown in FIG. 7 and FIG. 8. Sample No. 2, RSV-B positive, is shown in fig. 9. The primer group has good accuracy and high specificity.
It should be understood that the above-mentioned embodiments of the present invention are only examples for clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention, and it will be obvious to those skilled in the art that other variations or modifications may be made on the basis of the above description, and all embodiments may not be exhaustive, and all obvious variations or modifications may be included within the scope of the present invention.
Sequence listing
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Guangdong MudeYou Biotech Ltd
Primer combination, kit and detection method for mass spectrometry detection of multiple respiratory virus nucleic acids
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<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 62
ccatggtgga tggttggt 18
<210> 63
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 63
cgtgatgatg gtttcctgga 20
<210> 64
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 64
tataagcaca aagcacagag cgttcc 26
<210> 65
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 65
cagggaatct aagttcctcc ttgcca 26
<210> 66
<211> 16
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 66
ctgtgtactt ccttcg 16
<210> 67
<211> 17
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 67
tttcgtggta ttcttgc 17
<210> 68
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 68
ttgctttgct gctgcttg 18
<210> 69
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 69
cgcggagttg atcacaacta 20
<210> 70
<211> 15
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 70
ttgtcccgtg atctg 15
<210> 71
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 71
cagtagctgt ttctgaact 19
<210> 72
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 72
tgggaatttt agactaaaca g 21
Claims (8)
1. A primer combination for mass spectrometric detection of various respiratory viruses nucleic acids is characterized by comprising 48 amplification primers and 24 mass probe extension primers, wherein the nucleotide sequences of the amplification primers are shown as SEQ ID No. 1-48; the nucleotide sequence of the mass probe extension primer is shown in SEQ ID Nos. 49-72.
2. Use of the primer combination of claim 1 in the preparation of a plurality of respiratory virus nucleic acid mass spectrometric detection products.
3. A kit for mass spectrometric detection of multiple respiratory viral nucleic acids comprising the primer combination of claim 1.
4. The kit of claim 3, further comprising RT-PCR reaction reagents, dephosphorylation reaction reagents and mass probe extension reaction reagents.
5. A method for detecting a plurality of respiratory viruses using the kit of claim 3 or 4, comprising:
(1) carrying out RT-PCR amplification reaction on a sample to be detected by using 48 amplification primers;
(2) dephosphorizing the amplification product obtained in the step (1) by using alkaline phosphatase;
(3) performing single base extension on the dephosphorylated product obtained in the step (2) by using 24 mass probe extension primers;
(4) performing resin desalination and purification on the extension product obtained in the step (3);
(5) detecting by mass spectrum, and determining the pathogen type.
6. The method of claim 5, wherein the plurality of respiratory viruses are respiratory syncytial virus A and B, parainfluenza virus types 1 to 4, human metapneumovirus A and B, human bocavirus types 1 and 2, rhinovirus, influenza virus universal, influenza virus H1N1, influenza virus H3N2, influenza virus H1N12009, influenza virus B, coronavirus-SARS, coronavirus-MERS, novel coronavirus SARS-CoV-2, and adenovirus.
7. The method of claim 5, wherein the mass spectrometric detection is matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS).
8. The method of claim 5, wherein the alkaline phosphatase is shrimp alkaline phosphatase.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111286530A (en) * | 2019-12-27 | 2020-06-16 | 浙江迪谱诊断技术有限公司 | Primer group and kit for detecting 27 respiratory pathogens based on nucleic acid mass spectrometry and application of primer group and kit |
| CN111471804A (en) * | 2020-06-05 | 2020-07-31 | 浙江迪谱诊断技术有限公司 | Kit for detecting novel coronavirus with high sensitivity and high throughput and application thereof |
| CN111876524A (en) * | 2020-06-22 | 2020-11-03 | 江苏康为世纪生物科技有限公司 | Primer, probe combination and kit for detecting 34 respiratory pathogenic microorganisms based on multiple PCR-time-of-flight mass spectrometry |
| CN112899394A (en) * | 2020-11-23 | 2021-06-04 | 广州市达瑞生物技术股份有限公司 | Kit for detecting 33 respiratory pathogens by MALDI-TOF mass spectrometry platform |
-
2022
- 2022-01-21 CN CN202210074260.8A patent/CN114350857A/en active Pending
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111286530A (en) * | 2019-12-27 | 2020-06-16 | 浙江迪谱诊断技术有限公司 | Primer group and kit for detecting 27 respiratory pathogens based on nucleic acid mass spectrometry and application of primer group and kit |
| CN111471804A (en) * | 2020-06-05 | 2020-07-31 | 浙江迪谱诊断技术有限公司 | Kit for detecting novel coronavirus with high sensitivity and high throughput and application thereof |
| CN111876524A (en) * | 2020-06-22 | 2020-11-03 | 江苏康为世纪生物科技有限公司 | Primer, probe combination and kit for detecting 34 respiratory pathogenic microorganisms based on multiple PCR-time-of-flight mass spectrometry |
| CN112899394A (en) * | 2020-11-23 | 2021-06-04 | 广州市达瑞生物技术股份有限公司 | Kit for detecting 33 respiratory pathogens by MALDI-TOF mass spectrometry platform |
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