CN113755644A - Kit for detecting novel coronavirus Alpha and Delta mutant and application - Google Patents
Kit for detecting novel coronavirus Alpha and Delta mutant and application Download PDFInfo
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Abstract
The invention discloses a kit for rapidly detecting novel coronavirus Alpha and Delta mutants and application thereof, wherein the kit comprises a nucleotide sequence shown in SEQ ID NO: 1-2, and a second primer pair shown in SEQ ID NO.4-5, wherein the primer pair shown in SEQ ID NO: 3, a first probe as set forth in SEQ ID NO: 6, a host internal reference primer pair and a virus internal reference primer pair; the detection kit provided by the invention can be applied to sensitively and rapidly screening novel coronavirus Alpha and Delta mutants.
Description
Technical Field
The invention relates to the technical field of virus detection kits, in particular to a kit for detecting novel coronavirus Alpha and Delta mutant and application thereof.
Background
At present, mutants of the novel coronavirus SARS-CoV-2 belong to the B.1.1.7 (UK strain, Alpha) and B.1.617.2 (Indian strain, Delta) lineages, respectively, in the pango system and are called Alpha mutants and Delta mutants in the world-satellite tissue naming system. The Alpha mutants were reported to be most transmissible, with the Delta mutants being more lethal, second only to the Alpha mutants. The next generation high throughput sequencing technology (NGS) embodies its powerful performance in this detection of pandemic viral mutant genomes due to its ability to obtain the full-length genome of a new coronavirus. However, NGS sequencing requires reagents, consumables at a cost, and requires trained operators and skilled bioinformatic analysis capabilities. This allows NGS sequencing to still have short panels in this major epidemiological diagnosis.
Because the Alpha mutant and the Delta mutant cause serious wide spread in the world at present, how to rapidly and cheaply distinguish whether the two mutants are in SARS-CoV-2 positive samples is a very urgent need for timely adjusting the epidemic prevention.
The present invention concerns not only the detection of SARS-CoV-2 in clinical samples, but also whether the SARS-CoV-2 positive sample contains both mutants. Because the cost of the NGS sequencing technology is high, the variation can not be detected in real time, and the coverage of the sequence obtained by a sample with low nucleic acid content is low, the mutant strain type is difficult to accurately analyze.
Disclosure of Invention
The invention aims to provide a sensitive, quick and low-cost novel coronavirus Alpha and Delta mutant screening kit, and aims to excavate conservative characteristic mutations of Alpha and Delta mutants by combining bioinformatics analysis, thereby pertinently designing a primer probe and a kit for fluorescence quantitative PCR (polymerase chain reaction) capable of simultaneously detecting SARS-CoV-2 negative and positive mutants and whether the two mutants exist. The kit comprises 4 pairs of primer probes in total, and the detection is carried out in two reactions respectively. Provides technical support for screening the two high-transmission mutants.
The design key points of the mutant specific primers are that the conservative specific primers of the mutants are mined and designed through a bioinformatics algorithm. The primer can only amplify the nucleic acid containing the characteristic mutation carried by the mutant specifically and effectively. The characteristic mutation primers for specific targeted mutants need to ensure that the primers only match with the conservative characteristic mutations of the corresponding mutants. Due to the difference of the base sequence composition and spatial position and non-specific amplification, a characteristic mutation site cannot be randomly selected as a detection target. Primer design needs to be carried out on all conservative characteristic mutation sites of two mutants analyzed by bioinformatics, and the site with the best effect is selected as a target for distinguishing the corresponding mutants. Optimized to determine the best mutant detection primers and probes.
Specifically, the scheme of the invention is as follows:
a kit for detecting novel coronavirus Alpha and Delta mutants comprises a specific primer group and a fluorescent probe, wherein the specific primer group comprises a first primer pair, a second primer pair, a host internal reference primer pair and a virus internal reference primer pair, and the fluorescent probe comprises a first probe, a second probe, a host internal reference probe and a virus internal reference probe; the base sequence of the first primer pair is shown as SEQ ID NO: 1-2; the base sequence of the second primer pair is shown as SEQ ID NO. 4-5; the base sequence of the first probe is shown as SEQ ID NO: 3, the base sequence of the second probe is shown as SEQ ID NO: and 6.
According to the embodiment of the invention, the base sequence of the host internal reference primer pair is shown as SEQ ID NO: 7-8, wherein the base sequence of the virus internal reference primer pair is shown as SEQ ID NO: 10-11; the base sequence of the virus reference probe is shown as SEQ ID NO: 9, the base sequence of the virus reference probe is shown as SEQ ID NO: shown at 12.
According to the embodiment of the invention, the fluorescent reporter group of the kit fluorescent probe is FAM/HEX or HEX/ROX.
According to an embodiment of the invention, RT-PCR buffer, DNA polymerase, reverse transcriptase and RNase inhibitor are also included.
According to the embodiment of the invention, the final concentration of the specific primer group in the reaction system is 10pM, and the final concentration of the fluorescent probe in the reaction system is 5 pM.
According to an embodiment of the present invention, the kit further comprises a positive control prepared from a recombinant plasmid for detecting a gene.
The invention also provides the application of the kit in the preparation of SARS-CoV-2 detection products, and the detection products can detect SARS-CoV-2 and identify Alpha and Delta mutants at the same time.
Compared with the prior art, the invention has the beneficial effects that:
1. the detection kit provided by the invention can realize sensitive and rapid screening of novel coronavirus Alpha and Delta mutants, diagnosis of new crown infection by double RT-qPCR in two tubes and identification of infection strain Alpha and Delta mutants can be realized, and the sensitivity of 1copy/L can be achieved.
2. The kit provided by the invention selects the characteristic mutation site of A570D of Alpha mutant B.1.1.7 and the characteristic mutation site T19R of delta mutant B.1.617.2 as objects to design primer probes. The proportion of the two mutants which can detect the corresponding mutant strains is respectively 99.83 percent and 99.90 percent.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are required to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is a schematic diagram of the detection principle of the kit of the present invention.
FIG. 2 is a schematic diagram showing the common mutation conditions of the alpha mutant B.1.1.7 and delta mutant B.1.617.2 of SARS-CoV-2 of the present invention at the genome level.
Detailed Description
The following examples are intended to illustrate the invention without limiting its scope. It is intended that all modifications or alterations to the methods, procedures or conditions of the present invention be made without departing from the spirit and substance of the invention.
Therefore, the invention innovatively utilizes a bioinformatics method to analyze millions of public database SARS-CoV-2 genomes, excavates characteristic mutation information of Alpha and Delta mutants, designs fluorescent quantitative PCR primer probes for SARS-CoV-2 negative and positive detection and mutant detection according to the characteristic mutation information, and develops a kit for detecting the two mutants which are concerned at present.
The existing SARS-CoV-2 detecting reagent kit is aimed at several mutant sites of every mutant strain, and the detection sensitivity and specificity of these mutation detecting reagent kits are not good, and the detection cost of several sites is high. We have found the characteristic mutations of the Alpha and Delta mutants of SARS-CoV-2 by analytical comparison, and each mutant can be detected by using a mutation at one site.
The genomic analysis alignment of the SARS-CoV-2 mutant strain of one embodiment comprises:
the SARS-CoV-2 complete genome is first downloaded from the GISAID database. The downloaded whole genome is subjected to quality control by using self-compiled software, and genomes containing more than 5% of degenerate bases in the genome are filtered out (the high content of the degenerate bases means that the genomes are low in sequencing depth and low in coverage, and do not belong to high-quality genomes). The remaining genome was retained and considered as a high quality genome. SARS-CoV-2WIV04 (Genome Access: NC-045512.2) was used as the reference Genome. And carrying out pairwise global alignment on the downloaded high-quality genome and the reference genome respectively. Mutations, including point mutations, insertions and deletions (deletions) of the target sequence relative to the reference genome are extracted by an autonomously written mutation information extraction program. Meanwhile, a gene information annotation file (file format gff) of a reference genome is used as auxiliary information, and corresponding amino acid mutation is calculated according to a point mutation result of the whole genome and the gene annotation information. At the same time, downloading the pedigree information corresponding to each sequence of SARS-CoV-2 in the public database pango system. Combining the mutation information of each sequence with the pedigree information, calculating the mutation information of each pedigree. Then, to calculate common mutations per lineage, each mutation per lineage comprising more than 100 strains was calculated for the intra-lineage mutation frequency. The mutation frequency is calculated in the following manner: the frequency of a mutation of a certain lineage in that lineage is the number of sets of strains in which the mutation of a certain lineage co-occurs/the number of sets of all strains of a certain lineage. Mutations with a mutation frequency of greater than 50% were selected as common mutations for this lineage. After obtaining the information of the common mutation of each pedigree, the pedigree and the common mutation are respectively connected by using a graph theory model in mathematics. If a common mutation is only linked to a lineage, then the common mutation is considered to be characteristic of that lineage. If more than two mutations share the same common mutation, then the common mutation is linked to each of the two lineages. The relationship of pedigrees to common mutations was analyzed using the program written by the inventors. The results of the analysis were visualized using Cytoscape software. And directly screening characteristic mutations belonging to the Alpha mutant and the Delta mutant respectively according to a visualization result. As shown in FIG. 2, the common mutations at the genomic level of the Alpha mutant B.1.1.7 and the delta mutant B.1.617.2 of SARS-CoV-2 are shown. The characteristic mutations of the Alpha mutant B.1.1.7 and the delta mutant B.1.617.2 of SARS-CoV-2 are summarized and arranged to obtain three kinds of mutations of the two mutants shown in Table 1, wherein the characteristic mutation site of A570D of the Alpha mutant B.1.1.7 and the characteristic mutation site T19R of the delta mutant B.1.617.2 are selected as target design primer probes. The proportion of the two mutants which can detect the corresponding mutant strains is respectively 99.83 percent and 99.90 percent.
Table 1: results based on the genomic sequence of 1,240,000 SARS-CoV-2 on GSAID
The primer and Taqman probe sequence of the SARS-CoV-2 mutant alpha and Delta kit of one embodiment comprises:
a primer and TaqMan probe for rapidly and sensitively detecting novel coronavirus (SARS-CoV-2) Alpha and Delta mutants are characterized in that the base at the 3' end of an upstream primer of the primer is matched with the characteristic mutant base of the mutant and is mismatched with the base of a non-mutant, so that different efficiencies of qPCR amplification are caused, and whether the mutant exists is judged.
The primer probe consists of 4 pairs of primer probes, and comprises a specific primer probe for detecting Alpha and Delta mutants and a primer probe for detecting host internal reference (IC) and virus internal reference (SARS 2).
(1) Specific primer probes for detecting Alpha mutants:
Alpha-F：5’-CAACAATTTGGCAGAGACATTGT-3’(SEQ ID NO：1)
Alpha-R：5’-CCAAGTAGGAGTAAGTTGA-3’(SEQ ID NO：2)
Alpha-P：5’-HEX-TCCACAGACACTTGAGATTCTTGACA-BHQ1-3’(SEQ ID NO：3)
(2) specific primer probes for detecting Delta mutants:
Delta-F：5’-GTCAGTGTGTTAATCTTAT-3’(SEQ ID NO：4)
Delta-R：5’-GAACCAAGTAACATTGGAA-3’(SEQ ID NO：5)
Delta-P：5’-ROX-ACATTCAACTCAGGACTTGTTCTTACC-BHQ2-3’(SEQ ID NO：6)
(3) primer probe for detecting host gene:
IC-F：5’-AGTGCATGCTTATCTCTGACAG-3’(SEQ ID NO：7)
IC-R：5’-GCAGGGCTATAGACAAGTTCA-3’(SEQ ID NO：8)
IC-P：5’-HEX-TTTCCTGTGAAGGCG ATTGACCGA-BHQ1-3’(SEQ ID NO：9)
(4) conservative primer probe for detecting SARS-CoV-2:
SARS2-F：5’-CAATGGTTTAACAGGCACAGG-3’(SEQ ID NO：10)
SARS2-R：5’-CTCAAGTGTCTGTGGATCACG-3’(SEQ ID NO：11)
SARS2-P：5’-FAM-ACAGCATCAGTAGTGTCAGCAATGTCTC-BHQ1-3’ (SEQ ID NO：12)
the SARS-CoV-2 mutant alpha and Delta kit of one embodiment comprises:
the kit comprises the 4 sets of primers and probes, wherein the primers and probes for detecting human genes of coding ribonucleic acid P protein subunit P30(RPP30) respectively corresponding to IC, SARS, Alpha and Delta are used as human internal references, the primers and probes for conserved segments of RBD on novel coronavirus S genes are used as virus internal references, and Alpha mutant strains and Delta mutant strains of SARS-CoV-2. Two groups of primer probes for detecting virus internal reference and human internal reference are in one reaction, and two groups of primer probes for detecting two mutant strains are in the other reaction respectively. Further, the final working concentrations of the primers were all 0.4. mu.M, and the probe concentrations were all 0.1. mu.M. Further, the kit adopts a TaqMan real-time fluorescence quantitative PCR reaction system, the total system is 20 mu L, and the kit comprises 10 mu L Vazyme (Code No. Q222-01) Mix, 10 mu L Enzyme Mix, 0.8 mu L10 mu M upstream primer, 0.8 mu L10 mu M downstream primer, 0.2 mu L10 mu M probe, 2.2 mu L nuclease-free water and finally 5 mu L extracted RNA template. The reaction system is mixed evenly and then centrifuged for a short time, the reaction is carried out in BioRad CFX 96 fluorescence quantitative PCR, the reaction conditions are reverse transcription at 50 ℃ for 30min, pre-denaturation at 95 ℃ for 30s, amplification at 95 ℃ for 10s and 57 ℃ for 30s for 40 cycles, and fluorescence signals are collected.
The sensitivity of the kit is characterized by adopting RNA samples of Indian strain (Delta), English strain (Alpha) and Wuhan strain, the detection effect of the kit is evaluated, and the final result of the reverse transcription fluorescence quantitative PCR is shown in Table 2. It can be seen that the primer probe for detecting the mutant strain can effectively amplify the nucleic acid of the corresponding mutant strain, and the Ct value is very close to the Ct value of RBD conserved segment amplified by using the primer probe for virus internal reference, but the amplification efficiency of the non-target mutant strain or Wuhan strain is very low or no amplification is carried out, so that whether the mutant strain exists can be analyzed according to the difference of the Ct values.
Table 2: the detection results of 10-fold dilution, 100-fold dilution and 1000-fold dilution of each sample
The invention creatively utilizes the biogenic technology to analyze the high-quality sequencing data of large-scale SARS-CoV-2 genome in the GISAID database, excavates the conservative specific mutation sites of SARS-CoV-2Alpha and Delta mutant, combines an Amplification Retardation Mutation System (ARMS) with reverse transcription qPCR based on the two mutation sites, designs a primer probe and a kit for rapidly and sensitively detecting the two mutants, and greatly improves the detection efficiency and the detection speed of the mutant.
The sensitivity of the SARS-CoV-2 mutant alpha and Delta kit of one embodiment includes:
the sensitivity of the kit was further evaluated, and the sensitivity of the kit was measured by diluting the RNA of Indian and British strains in a gradient manner. The Ct values of the fluorescent quantitative PCR as shown in Table 3 were obtained. The copy number corresponding to the lowest sample concentration which can be detected is the detection limit for detecting the mutant strain. As a result, the lowest detection limit of the kit is 1 copy/. mu.L.
Table 3: detection result of sensitivity of kit to Delta and alpha
The application of the SARS-CoV-2 mutant alpha and Delta kit in clinical sample detection includes:
the kit is used for detecting 80 clinical samples, and the detection results are shown in table 4. Out of 80 clinical samples, 2Alpha mutants of SARS-CoV-2, 35 Delta mutants of SARS-CoV-2, 43 non-SARS-CoV-2 Alpha mutants, non-SARS-CoV-2 Delta mutants, or negative samples were detected. These mutants were verified by NGS or generation sequencing. As can be seen from the results in Table 4, for samples with very low nucleic acid content (above Ct value 38), the kit can also be used to detect whether the target mutant strain is the target mutant strain, which indicates the sensitivity and specificity of the kit. For some samples with high nucleic acid content (Ct value <29), some mutant strains can detect higher Ct value (>35), and for these samples with high nucleic acid content, the kit has weak non-specific amplification, so in the practical implementation process, the difference between the Ct value detected by the mutant strain and the Ct value detected by the virus (SARS2) is more than 5, which indicates that no target mutant strain exists.
Table 4: the kit is applied to the identification result of SARS-CoV-2 mutant in 80 clinical samples
The invention is not limited solely to that described in the specification and embodiments, and additional advantages and modifications will readily occur to those skilled in the art, so that the invention is not limited to the specific details, representative apparatus, and illustrative examples shown and described herein, without departing from the spirit and scope of the general concept as defined by the appended claims and their equivalents.
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Claims (7)
1. A kit for detecting novel coronavirus Alpha and Delta mutants is characterized by comprising a specific primer group and a fluorescent probe, wherein the specific primer group comprises a first primer pair, a second primer pair, a host reference primer pair and a virus reference primer pair; the base sequence of the first primer pair is shown as SEQ ID NO: 1-2; the base sequence of the second primer pair is shown as SEQ ID NO. 4-5; the base sequence of the first probe is shown as SEQ ID NO: 3, the base sequence of the second probe is shown as SEQ ID NO: and 6.
2. The kit according to claim 1, wherein the base sequence of the host internal reference primer pair is as shown in SEQ ID NO: 7-8, wherein the base sequence of the virus internal reference primer pair is shown as SEQ ID NO: 10-11; the base sequence of the virus internal reference probe is shown as SEQ ID NO: 9, the base sequence of the virus reference probe is shown as SEQ ID NO: shown at 12.
3. The kit of claim 1, wherein the fluorescent reporter group of the kit fluorescent probe is FAM/HEX or HEX/ROX.
4. The kit of claim 1, further comprising RT-PCR buffer, DNA polymerase, reverse transcriptase, and rnase inhibitor.
5. The kit according to claim 1, wherein the final concentration of the specific primer group in the reaction system is 10pM, and the final concentration of the fluorescent probe in the reaction system is 5 pM.
6. The kit of claim 1, further comprising a positive control prepared from a recombinant plasmid for detecting a gene.
7. Use of the kit of any one of claims 1 to 6 for the preparation of a SARS-CoV-2 assay product which can detect SARS-CoV-2 and identify both Alpha and Delta mutants.
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