CN114686620B - Novel primer combination, kit and detection method for detecting nucleic acid mass spectrum of various variants of coronaviruses - Google Patents

Novel primer combination, kit and detection method for detecting nucleic acid mass spectrum of various variants of coronaviruses Download PDF

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CN114686620B
CN114686620B CN202210074264.6A CN202210074264A CN114686620B CN 114686620 B CN114686620 B CN 114686620B CN 202210074264 A CN202210074264 A CN 202210074264A CN 114686620 B CN114686620 B CN 114686620B
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artificial sequence
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detection
primer
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CN114686620A (en
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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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Bioisland Laboratory
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Abstract

The invention discloses a primer combination, a kit and a detection method for detecting nucleic acid mass spectra of a plurality of variants of novel coronaviruses, and relates to the technical field of molecular biology detection. The kit comprises 16 amplification primers and 9 mass probe extension primers, and can further perform typing identification on various variant strains while detecting novel coronaviruses through the combination of 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 detecting nucleic acid mass spectrum of various variants of coronaviruses
Technical Field
The invention relates to the technical field of molecular biology detection. More particularly, it relates to a primer combination, a kit and a detection method for detecting nucleic acid mass spectra of a plurality of variants of novel coronaviruses.
Background
For the nucleic acid detection of new coronaviruses, the main current method is the fluorescent quantitative RT-PCR method. Due to the limitation of fluorescent channels, only two targets of N and ORF1ab of the new coronavirus can be detected at a time, and effective typing cannot be carried out, and particularly under the condition of low viral load, single positive and false negative conditions are easy to occur. NGS high-throughput sequencing is also an important technology for detecting new coronaviruses, and accurate identification of viruses and subtypes can be realized through full sequencing, but the whole flow is long in time, and the virus detection result cannot be rapidly given; and the experimental operation is complex, and the sample detection cost is high. In the case of large-area explosion of epidemic situations, the method can be used for rapidly and accurately detecting and diagnosing an infected person or a carrier, and has a vital effect and significance for epidemic situation prevention and control, so that a detection method with high specificity, high sensitivity, high flux and relatively simple operation and low cost is urgently needed for detecting, identifying and further typing new coronavirus variant strains.
Disclosure of Invention
The invention aims to provide a primer combination for detecting the nucleic acid mass spectrum of a plurality of variants of novel coronaviruses and a detection kit containing the primer combination, and the screening and diagnosis capability of the novel coronaviruses is improved.
The invention also aims at providing a method for detecting the nucleic acid mass spectrum of the novel coronavirus multiple variant strain by using the kit. The method ensures that the novel coronavirus has strong detection specificity and high sensitivity.
In order to achieve the above purpose, the invention adopts the following technical scheme:
In a first aspect, the invention provides a primer combination for detecting nucleic acid mass spectra of a plurality of variants of novel coronaviruses, which comprises 16 amplification primers and 9 mass probe extension primers, wherein the nucleotide sequences of the amplification primers are shown in SEQ ID No.1-16, and the specific nucleotide sequences are shown in Table 1; the nucleotide sequence of the mass probe extension primer is shown in SEQ ID Nos. 17-25, 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 the MALDI-TOF mass spectrometry technology, namely, PCR-micro sequencing is adopted to detect novel coronavirus nucleic acid and the typing of a plurality of variants. In the invention, a series of PCR amplification primers and quality probe extension (MPE) primers for detecting novel coronavirus variant strains are designed aiming at selected detection target genes and specific mutation sites by carrying out sequence analysis on the novel coronavirus variant strains. Wherein, the invention selects novel coronavirus genes N, ORF1ab, S-D614G as general target genes, and uses at least two gene detections as positive judgment standard of the novel coronavirus; meanwhile, the specific mutation sites of various novel coronavirus variants are selected as parting detection targets, so that parting detection of the novel coronavirus variants is realized.
The invention also provides application of the primer combination in preparing novel coronavirus multiple variant nucleic acid mass spectrum detection products.
According to the specific embodiment of the invention, the invention provides a novel coronavirus multiple variant nucleic acid mass spectrometry detection kit, which comprises the primer combination.
Further, 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 nucleic acid mass spectra of multiple variants of coronavirus, the method using the kit, specifically comprising:
(1) Carrying out RT-PCR amplification reaction on a sample to be detected by using 16 amplification primers;
(2) Dephosphorylation of the amplified product obtained in step (1) with alkaline phosphatase;
(3) Extending the primer pair by using 9 mass probes to carry out single base extension on the dephosphorylated product obtained in the step (2);
(4) Carrying out resin desalination and purification on the extension product obtained in the step (3);
(5) And (5) mass spectrum detection, and determining variant strain typing.
Further, the novel coronavirus multiple variants are Alpha variants, beta variants, delta variants or Lambda variants.
Further, the mass spectrum detection adopts matrix-assisted laser desorption ionization time-of-flight mass spectrometry MALDI-TOF MS.
Further, the alkaline phosphatase is shrimp alkaline phosphatase.
The beneficial effects of the invention are as follows:
The invention combines the multiplex PCR technology and the MALDI-TOF technology, adopts the PCR-MALDI micro-sequencing method, and can realize the detection of novel coronaviruses and the further typing identification of four variants by using the specific primer combination of the invention. Wherein, part of the 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 detection limit of the sample is 10 copies/. Mu.L, the false positive result can be obviously reduced, and the detection and identification of the low-load virus sample can be improved. The detection flux is high, 96/384 samples can be detected each time, and the method is more suitable for large-scale screening of new coronavirus cases.
Drawings
The following describes the embodiments of the present invention in further detail with reference to the drawings.
FIG. 1 shows the results of the specificity verification of multiplex PCR amplification primers and MPE primers.
FIG. 2 shows the amplification spectra of the positive plasmids from the precision experiments.
FIG. 3 shows the Delta site positive mass spectrum of 202101 samples.
FIG. 4 shows a Delta site negative mass spectrum of 202102 samples.
FIG. 5 shows a positive mass spectrum of the novel crown ORF1ab site of 202102 samples.
FIG. 6 shows a Delta site positive mass spectrum of 202103 samples.
FIG. 7 shows a Delta site negative mass spectrum of 202104 samples.
FIG. 8 shows a Delta site positive mass spectrum of 202105 samples.
Detailed Description
In order to more clearly illustrate the present invention, the present invention will be further described with reference to preferred embodiments and the accompanying drawings. Like parts in the drawings 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 that this invention is not limited to the details given herein.
Reagents and instrumentation used in the course of the experiment:
VIRAL RNA MINI KIT (52904): kaijia corporation manages (Shanghai) limited;
nucleic acid detection kit (pathogenic microorganism) (QT-SJ 12-RTs): intellectual development biotechnology (Qingdao) limited; the kit comprises:
and A1: RT-PCR enzyme, A2 component: RT-PCR buffer;
And A3: SAP enzyme, A4 component: SAP buffer;
And A5: MPE enzyme, A6 component: MPE buffer, A7 component: e_ ddNTPmix;
And A8: a matrix liquid;
plasmid and primer synthesis: bioengineering (Shanghai) stock Co.Ltd;
veriti TM 96 well thermal cycler: siemens Feishul technologies;
NanoDrop TM One spectrophotometer: siemens Feishul technologies;
QuanTOF I mass spectrometer: intellectual development biotechnology (Qingdao) limited.
Example 1 amplification primer and quality probe extension primer design
The complete sequences of 4 new crown variants were downloaded from NCBI, 5-10 strains each were downloaded, aligned using BioEdit sequence Alignment Editor software, key genes such as N, ORF1ab, S, etc. of SARS-CoV-2 were aligned, intraspecies conservation was selected, interspecific specific target genes were detected (see Table 3), and human RNaseP was selected as an internal reference. Multiplex PCR Primer design was performed using Primer3 online webpage. Wherein adjacent nearer sites share a pair of PCR primers. Specific amplification primers are shown in Table 1 above. The mutation sites were selected and Mass Probe Extension (MPE) primer design was performed using genetic locus typing system software (intellectualized biotechnology (peninsula)) as shown in table 2 above. In particular, for Beta variant detection, a new primer sequence design strategy is adopted, and the last base at the 3' -end is designed to be the specific site of the Beta variant, so that the primer specificity is ensured. If the mutant exists in the sample, the base A is extended, otherwise, no extension exists, and the detection result is Nocall. The PCR primers and MPE primers were designed and plasmid synthesis and primer synthesis were performed by the division of Biotechnology (Shanghai) Co., ltd.
TABLE 3 SARS-CoV-2 selection of target genes
Pathogen name Target gene
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 set-up
1. And (5) diluting the plasmid.
The plasmid dry powder obtained in example 1 was diluted with water at a concentration of 100 ng/. Mu.L and accurately quantified using Nanodrop. The number of copies contained in the plasmid was calculated based on the plasmid sequence. The concentrations used in the experiments were varied, respectively 105copies/μL,104copies/μL,103copies/μL,102copies/μL,101copies/μL,100copy/μL.
2. Primer dilution and mixing.
Preparing a PCR primer mixture: after the PCR primer sequences were synthesized, the dry powder was dissolved in water to 100. Mu.M stock solution. The stock solution was taken out and mixed to prepare a primer mixture having a final concentration of the primer at each site in the range of 0.5. Mu.M to 5. Mu.M.
Preparing MPE primer mixed solution: after mass probe extension primer sequences were synthesized, the dry powder was dissolved in water to 500. Mu.M stock solution. The primer stock was taken out and mixed to prepare a primer mixture having a final concentration of primer at each site in the range of 5. Mu.M to 15. Mu.M.
RT-PCR reactions
(1) The RT-PCR system was configured using a nucleic acid detection kit (pathogenic microorganism) (QT-SJ 12-RTs), as shown in Table 4.
TABLE 4 RT-PCR System configuration Table
Reagent component Volume (mu L)
RT-PCR enzyme 1
RT-PCR premix 12.5
PCR primer mixture 5
Template 6.5
Total 25
(2) The RT-PCR procedure was run as in Table 5.
TABLE 5 RT-PCR temperature control reaction procedure
(3) After the reaction was completed, 5. Mu.L of the amplified product was taken out for subsequent experimental reaction.
4. Shrimp Alkaline Phosphatase (SAP) dephosphorylation treatment
(1) The SAP reaction system was configured using a nucleic acid detection kit (pathogenic microorganisms) (QT-SJ 12-RTs). SAP reaction solutions were prepared as shown in table 6.
Table 6 SAP architecture configuration table
Reagent component Volume (mu L)
SAP enzyme 0.3
SAP buffer 0.17
H2O 1.53
Total 2
Table 6 SAP architecture configuration table
(2) Mu.L of SAP reaction solution was added to 5. Mu.L of the amplification product removed in the previous step, and the mixture was placed on a PCR instrument to run SAP dephosphorylation reaction.
Table 7 SAP temperature controlled reaction program
Reaction temperature Time of
37℃ 40min
85℃ 5min
4℃ hold
MPE quality Probe extension
(1) The MPE reaction system was configured using a nucleic acid detection kit (pathogenic microorganism) (QT-SJ 12-RTs). MPE reaction solutions were prepared as in Table 8.
Table 8 MPE architecture configuration table
Reagent component Volume (mu L)
MPE enzyme 0.6
MPE buffer 1.4
E_ddNTPmix 1
MPE primer mixture 1
Total 4
(2) Mu.L of MPE reaction solution was added to 7. Mu.L of the dephosphorylated product of the previous step, and the mixture was placed on a PCR instrument to run the MPE reaction.
Table 9 MPE temperature control program
6. Resin desalting and purifying, and target plate spotting.
(1) To each reaction well was added 14 μl deionized water.
(2) The eight-joint tube with the resin is gently turned over and buckled on the sample plate, so that the alignment of the resin holes and each hole of the sample is ensured. The resin tube is then tapped to drop the resin into the wells of the sample plate.
(3) The sample plate with the resin was placed in a tumble mixer and mixed for 30min at 20 rpm.
(4) After the completion of the 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) 1. Mu.L of the mixed solution was spotted on a target plate.
7. Target plate acquisition and data analysis.
(1) According to QuanTOF instrument instructions, the matrix and sample co-crystallized target plate is loaded into the instrument, and data acquisition is carried out after vacuum degree reaches the requirement (BA Gauge is better than 2e-6 Torr). The acquisition mode of the instrument is a linear positive ion mode, and important parameters are set as follows: ACCELERATE VOLTAGE:20kV, MASS RANGE:3000-11000Da, laser frequency:3000Hz, shots/spectrum:800, laser energy:24 uJ.
(2) After the collection is completed, clicking analysis is performed, software can give the extension of each site of each sample, and the detection result of the sample can be checked. Important parameters are set as follows: SNR:4.0.
Example 3 specificity experiments
1. Primer combinations template-free amplification verifies primer specificity. Firstly, water is used for replacing a sample template, see the whole experimental procedure from the RT-PCR reaction to resin desalination and purification in the example 2, and the step is to verify that no dimer exists between the multiplex PCR amplification primer and the multiplex MPE primer, no non-specific amplification extension product exists, and the specificity of primer design is ensured. The experimental results are shown in FIG. 1.
2. Other pathogens were used to verify primer specificity. Other viruses (e.g., a-stream, b-stream, etc.), bacterial mycoplasma pneumoniae, chlamydia, etc., are also common to cause respiratory tract infections. The experiment uses a Flu A sample, an ADV sample and mycoplasma pneumoniae, and the verification test shows that no new crown target is detected, so that other pathogenic microorganisms are ensured not to report incorrect detection results in a non-specific way.
Example 4 sensitivity and precision experiments
(1) Sensitivity verification results:
The plasmid dry powders of the examples were subjected to 10-fold concentration gradient dilutions of 105 copies/. Mu.L, 104 copies/. Mu.L, 103 copies/. Mu.L, 102 copies/. Mu.L, 101 copies/. Mu.L and 100 copies/. Mu.L, respectively, using water. Six concentrations of plasmid template were amplified and extended, respectively. The end result is a novel coronavirus with a lower detection limit of 10 copies/. Mu.L. See in particular table 10.
TABLE 10 detection sites for novel coronaviruses and lower limit of detection of mutant sites
(2) Precision verification result:
using plasmid concentration 100 copies/. Mu.L, 3 replicates per experiment, 6 replicates were completed 2 weeks in total, 9 products could be detected, and the detection results were stable. See fig. 2.
Example 5 sample detection
5 Quality samples were obtained for the new coronavirus delta variant nucleic acid detection chamber interstitial assessment. Nucleic acid extraction was performed using the Kanji virus extraction kit (VIRAL RNA MINI KIT (52904)), and novel coronavirus nucleic acid mass spectrometry was performed according to the method of example 2.
The results are shown in Table 11 and FIGS. 3-8, with 4 positive samples successfully detected. The primer group has good accuracy and high specificity.
Table 11 test results of 5 quality samples
It should be understood that the foregoing examples of the present invention are provided for illustration only and are not intended to limit the embodiments of the present invention, and that various other changes and modifications can be made by those skilled in the art based on the foregoing description, and it is not intended to be exhaustive of all the embodiments, and obvious changes and modifications that come within the scope of the invention are defined by the appended claims.
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Claims (4)

1. The primer combination for detecting the novel coronavirus multiple variant nucleic acid mass spectrum is characterized by comprising 16 amplification primers and 9 mass probe extension primers, wherein the nucleotide sequence of the amplification primers is shown as SEQ ID No. 1-16; the nucleotide sequence of the mass probe extension primer is shown in SEQ ID No. 17-25; the novel coronavirus multiple variants are Alpha variants, beta variants, delta variants or Lambda variants.
2. The use of the primer combination of claim 1 for preparing a novel coronavirus multi-variant nucleic acid mass spectrometry detection product.
3. A novel coronavirus multiple variant nucleic acid mass spectrometry detection kit comprising the primer combination of claim 1.
4. The kit of claim 3, further comprising an RT-PCR reaction reagent, a dephosphorylation reaction reagent, and a mass probe extension reaction reagent.
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