CN114686620A - Novel primer combination, kit and detection method for mass spectrometric detection of multiple variant strains of coronavirus - Google Patents

Novel primer combination, kit and detection method for mass spectrometric detection of multiple variant strains of coronavirus Download PDF

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CN114686620A
CN114686620A CN202210074264.6A CN202210074264A CN114686620A CN 114686620 A CN114686620 A CN 114686620A CN 202210074264 A CN202210074264 A CN 202210074264A CN 114686620 A CN114686620 A CN 114686620A
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翟志向
周晓光
宋合兴
李晨
李运涛
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Rongzhi Biotechnology Qingdao Co ltd
Bioisland Laboratory
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Abstract

The invention discloses a primer combination, a kit and a detection method for mass spectrometric detection of various variants of a novel coronavirus, and relates to the technical field of molecular biology detection. The kit comprises 16 amplification primers and 9 mass probe extension primers, and can detect the novel coronavirus and further classify and identify various variants 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 screening of new coronavirus cases.

Description

Novel primer combination, kit and detection method for mass spectrometric detection of multiple variant strains of coronavirus
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 spectrometric detection of multiple variants of a novel coronavirus.
Background
For the nucleic acid detection of the novel coronavirus, the current main method is a fluorescent quantitative RT-PCR method. Due to the limitation of a fluorescent channel, only two targets of N, ORF1ab can be detected for the new coronavirus at one time, and effective typing cannot be performed, particularly under the condition of low viral load, the situations of single positive and false negative easily occur. NGS high-throughput sequencing is also an important technology for detecting new coronavirus, and accurate identification of virus and subtype can be realized through full sequencing, but the whole process is long in time, and a virus detection result cannot be quickly given; and the experiment operation is complex, and the sample detection cost is high. When the epidemic situation explodes in a large area, the method can quickly and accurately detect and diagnose infected persons or carriers and has a vital role and significance for epidemic situation prevention and control, so that a low-cost detection method which is high in specificity, high in sensitivity, high in flux and relatively simple in operation is urgently needed for detecting, identifying and further typing the new coronavirus variant.
Disclosure of Invention
The invention aims to provide a primer combination for mass spectrometric detection of multiple variants of a novel coronavirus and a detection kit containing the primer combination, so that the screening and diagnosis capabilities of the novel coronavirus are improved.
Another object of the present invention is to provide a method for performing nucleic acid profiling of various variants of the novel coronavirus using the kit. The method has the advantages of strong specificity and high sensitivity of the novel coronavirus 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 variants of a novel coronavirus, comprising 16 amplification primers and 9 mass probe extension primers, wherein the nucleotide sequences of the amplification primers are shown as SEQ ID Nos. 1-16, and are specifically shown in Table 1; the nucleotide sequence of the mass probe extension primer is shown in SEQ ID No.17-25, and is specifically shown in Table 2.
TABLE 1 amplification primer sequences
Figure BDA0003483357570000011
Figure BDA0003483357570000021
TABLE 2 Mass Probe Extension (MPE) primer sequences
Figure BDA0003483357570000022
The invention combines the multiplex PCR technology with MALDI-TOF mass spectrum technology, namely PCR-micro sequencing is adopted to carry out novel coronavirus nucleic acid detection and typing detection of a plurality of variants. In the invention, a series of PCR amplification primers and quality probe extension (MPE) primers for detecting the novel coronavirus variant strain are designed aiming at a selected target gene and a specific mutation site through sequence analysis of a plurality of variant strains of the novel coronavirus. The method selects new coronavirus genes N, ORF1ab and S-D614G as universal target genes, and detects at least two genes as a positive judgment standard of the new coronavirus; meanwhile, specific mutation sites of various novel coronavirus variant strains are selected as typing detection targets, and the typing detection of the novel coronavirus variant strains is realized.
The invention also provides application of the primer combination in preparing a novel coronavirus multiple variant nucleic acid mass spectrum detection product.
According to the specific embodiment of the invention, the invention provides a novel nucleic acid mass spectrometry detection kit for multiple variants of coronavirus, 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 novel method for detecting multiple variants of coronavirus by nucleic acid mass spectrometry, wherein the method uses the kit, and specifically comprises:
(1) carrying out RT-PCR amplification reaction on a sample to be detected by utilizing 16 amplification primers;
(2) dephosphorizing the amplification product obtained in the step (1) by using alkaline phosphatase;
(3) carrying out single base extension on the dephosphorylated product obtained in the step (2) by utilizing 9 mass probe extension primers;
(4) performing resin desalination and purification on the extension product obtained in the step (3);
(5) and (5) detecting by mass spectrum, and determining the type of the variant strain.
Furthermore, the various variants of the novel coronavirus are an Alpha variant, a Beta variant, a Delta variant or a Lambda variant.
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 combines the multiplex PCR technology and the MALDI-TOF technology, adopts the PCR-MALDI micro-sequencing method, and uses the specific primer combination of the invention, so that the novel coronavirus can be detected, and the four variants can be further typed and identified. Wherein, partial PCR primers of the detection sites are shared, thereby reducing the synthesis cost of the primers. On the detection result, the accuracy is good, the specificity is strong, the sensitivity is high, the sample detection limit is 10 copies/mu L, the false positive result can be obviously reduced, and the detection and identification of the low-load virus sample are improved. The detection flux is high, 96/384 samples can be detected each time, and the kit is more suitable for large-scale screening of new coronavirus cases.
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The following describes embodiments of the present invention in further detail with reference to the accompanying drawings.
Figure 1 shows the specificity validation results for multiplex PCR amplification primers and MPE primers.
FIG. 2 shows the amplified mass spectrum of the positive plasmid for the precision experiment.
FIG. 3 shows a mass spectrum positive for the Delta site of the 202101 sample.
FIG. 4 shows a Delta site negative mass spectrum of the 202102 sample.
FIG. 5 shows a mass spectrum positive for the neocorona ORF1ab site from the 202102 sample.
FIG. 6 shows a mass spectrum positive for the Delta site of the 202103 sample.
FIG. 7 shows a Delta site negative mass spectrum of the 202104 sample.
FIG. 8 shows a mass spectrum positive for the Delta site of the 202105 sample.
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 components 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) Inc.;
nucleic acid detection kit (pathogenic microorganism) (QT-SJ 12-RTs): science and technology (Qingdao) Inc.; the kit comprises:
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 strains of 4 new crown variant strains are downloaded from NCBI, 5-10 strains are downloaded for each strain, the BioEdit sequence Alignment Editor software is used for sequence comparison, key genes such as N, ORF1ab, S and the like of SARS-CoV-2 are mainly compared, target genes with intra-species conservation and inter-species specificity are selected for detection (see table 3), and human RNaseP is selected as an internal reference. Multiplex PCR Primer design was performed using Primer3 on-line web pages. Wherein the adjacent and closer sites share a pair of PCR primers. 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. Particularly, for Beta variant strain detection, a new primer sequence design strategy is adopted, and the last base at the 3' end is designed to be a specific site of the Beta variant strain, so that the primer specificity is ensured. If the mutant strain exists in the sample, the base A is extended, otherwise, the extension is not performed, and the detection result is Nocall. The designed PCR primers and MPE primers were synthesized by engineering bioengineering (Shanghai) Inc. for plasmid and primer synthesis, respectively.
TABLE 3 SARS-CoV-2 selection target genes
Pathogen name Target genes
SARS-CoV-2 N
SARS-CoV-2 ORF1ab
SARS-CoV-2-S-D614G S
Alpha S
Beta S
Delta S
Lambda S
Example 2 detection method establishment
1. And (5) diluting the plasmid.
The dry plasmid powder obtained in example 1 was diluted with water to a concentration of 100 ng/. mu.L 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 PCR primer sequences 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 were synthesized, the dry powder was dissolved in 500. mu.M stock solution using water. And taking out the primer stock solution, and mixing to prepare a primer mixture solution with the final concentration of the primer at 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 components Volume (μ L)
RT-PCR enzymes 1
RT-PCR premix solution 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
Figure BDA0003483357570000051
(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
Reagent composition Volume (μ L)
SAP enzymes 0.3
SAP buffer 0.17
H2O 1.53
Total 2
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 and run MPE reaction.
TABLE 9 MPE temp. CONTROL PROGRAM
Figure BDA0003483357570000061
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 octant tubes were gently inverted and snapped onto the sample plate to ensure that the resin wells were aligned with each well of 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 with a co-crystallized substrate and a 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. Other pathogens were used to verify primer specificity. Other viruses (e.g., influenza A, influenza B, etc.), bacterial Mycoplasma pneumoniae, Chlamydia, etc., are also common in causing respiratory tract infections. The Flu A sample, the ADV sample and the mycoplasma pneumoniae are used in the experiment, and the verification test proves that no new corona target is detected, so that the false detection result cannot be reported in a non-specific manner by other pathogenic microorganisms.
Example 4 sensitivity and precision experiments
(1) The result of the sensitivity verification:
the dry plasmid powder of the examples was diluted with water in 10-fold concentration gradients of 105 copies/. mu.L, 104 copies/. mu.L, 103 copies/. mu.L, 102 copies/. mu.L, 101 copies/. mu.L, and 100 copy/. mu.L, respectively. Six concentrations of plasmid template were amplified and extended, respectively. The end result was that the lower limit of detection of the novel coronavirus was 10 copies/. mu.L. See table 10 for details.
TABLE 10 detection lower limits of New coronavirus detection sites and variant sites
Figure BDA0003483357570000071
Figure BDA0003483357570000081
(2) And precision verification results are as follows:
the plasmid concentration is 100 copies/mu L, 3 times of repeated experiments are carried out in each experiment, 6 repeated experiments are completed in 2 weeks, 9 products can be detected, and the detection result is stable. See fig. 2.
EXAMPLE 5 sample testing
Participating in the nucleic acid detection of new coronavirus delta variant strain for indoor evaluation, and obtaining 5 quality evaluation samples. Nucleic acid extraction was performed using a Qiagen virus extraction Kit (Viral RNA Mini Kit (52904)), and mass spectrometric detection of novel coronavirus nucleic acids was performed according to the method of example 2.
The results are shown in Table 11 and FIGS. 3-8, and 4 groups of positive samples were successfully detected. The primer group has good accuracy and high specificity.
Test results of 115 quality evaluation samples in Table
Figure BDA0003483357570000082
It should be understood that the above-described embodiments of the present invention are merely examples for clearly illustrating the invention and are not intended to limit the embodiments of the present invention, and that various other modifications and variations can be made on the basis of the above-described embodiments by those skilled in the art.
Sequence listing
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Primer combination, kit and detection method for mass spectrometric detection of multiple variant strains of novel coronavirus
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Claims (8)

1. A primer combination for mass spectrometric detection of various variants of a novel coronavirus is characterized by comprising 16 amplification primers and 9 mass probe extension primers, wherein the nucleotide sequences of the amplification primers are shown as SEQ ID Nos. 1-16; the nucleotide sequence of the mass probe extension primer is shown in SEQ ID No. 17-25.
2. Use of the primer combination of claim 1 in the preparation of a novel product for mass spectrometric detection of multiple variant nucleic acids of coronaviruses.
3. A novel kit for detecting multiple variants of coronavirus by nucleic acid mass spectrometry, which comprises 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 novel method for detecting multiple variants of coronavirus through nucleic acid mass spectrometry, which is characterized by using the kit as claimed in claim 3 or 4, and comprises the following steps:
(1) carrying out RT-PCR amplification reaction on a sample to be detected by utilizing 16 amplification primers;
(2) dephosphorizing the amplification product obtained in the step (1) by using alkaline phosphatase;
(3) carrying out single base extension on the dephosphorylated product obtained in the step (2) by utilizing 9 mass probe extension primers;
(4) performing resin desalination and purification on the extension product obtained in the step (3);
(5) and (5) detecting by mass spectrum, and determining the type of the variant strain.
6. The method of claim 5, wherein the variant of the novel coronavirus is an Alpha variant, a Beta variant, a Delta variant, or a Lambda variant.
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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