CN114107574A - Kit and method for detecting novel coronavirus and Omicron mutant strain thereof - Google Patents
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Abstract
The invention discloses a kit and a method for detecting novel coronavirus and an Omicron mutant strain thereof. The invention carries out qualitative detection on the novel coronavirus by carrying out combined detection on three target genes of new crown ORF1ab and N, S, and simultaneously, the S gene detection target region covers the specific mutation site of the Omicron variant, if the novel coronavirus is the Omicron variant, S gene loss is caused to have no signal due to the occurrence of the mutation site, so that the Omicron variant and non-Omicron variants are identified. The detection reagent provided by the invention can be used for detecting the novel coronavirus and the patient infected by the Omicron mutant strain thereof with high efficiency, high specificity, high stability and low cost.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a kit and a method for detecting novel coronavirus and an Omicron mutant strain thereof.
Background
The novel coronavirus (SARS-CoV-2) is a single-stranded positive-strand RNA virus and is susceptible to mutation. Clinically, the symptoms of patients infected with the new coronavirus are greatly different, and the patients are likely to have symptoms from asymptomatic to critical. In addition to differences in individual factors, viral variation may also be a significant factor in the wide variation of symptoms in infected patients.
Research shows that the mutation rate of the novel coronavirus is about 2-4 mutations per month, and variant strains with more than 25 mutations are found in known strains at present. Alpha new crown variant strain, Beta new crown variant strain, Gamma new crown variant strain, Delta new crown variant strain and Omicron new crown variant strain are discovered in a plurality of countries successively, and the Alpha new crown variant strain, the Beta new crown variant strain, the Delta new crown variant strain and the Omicron new crown variant strain are mutated in a Receptor Binding Domain (RBD) of a virus surface spike protein, so that the Alpha new crown variant strain and the Delta new crown variant strain are more easily combined with an angiotensin converting enzyme 2(ACE2) receptor on the surface of a human cell, and the transmission capability and the pathogenic capability of the virus can be obviously influenced by the variation of the virus.
The new coronavirus Ormcken variant has more mutations in virus Spike (Spike) protein, and has important amino acid mutation sites of the first 4 variants, namely Alpha (Alpha), Beta (Beta), Gamma (Gamma) and Delta (Delta) Spike protein, including mutation sites for enhancing cell receptor affinity and virus replication capacity, so that the new coronavirus Ormcken variant has extremely strong transmission capacity.
Therefore, the development of a combined screening kit capable of simultaneously detecting the novel coronavirus and the Omicron variant strain has a very positive significance for epidemic prevention and control and treatment of patients with confirmed diagnosis.
Disclosure of Invention
The invention develops a composition, a kit, a method and application for rapidly detecting novel coronavirus and an Omicron mutant strain aiming at the novel coronavirus and the Omicron mutant strain thereof.
In a first aspect of the present invention, there is provided a set of primer pairs for detecting a novel coronavirus and its Omicron variant strain, the set of primer pairs comprising a first primer pair:
the first primer pair comprises: a forward primer shown as SEQ ID NO. 7; and, a reverse primer as shown in SEQ ID NO. 8.
In another preferred embodiment, the primer pair set further comprises other primer pairs for detecting the novel coronavirus.
In another preferred example, the primer pair set further comprises a primer pair for detecting the new coronavirus ORF1ab gene, and/or N gene.
In another preferred example, the primer pair set further comprises a second primer pair that specifically amplifies a novel coronavirus ORF1ab gene, the second primer pair comprising:
a forward primer shown as SEQ ID NO. 1; and, a reverse primer as shown in SEQ ID NO. 2.
In another preferred example, the primer pair set further comprises a third primer pair, the third primer pair specifically amplifies a novel coronavirus N gene, and the third primer pair comprises:
a forward primer shown as SEQ ID NO. 4; and, a reverse primer as shown in SEQ ID NO. 5.
In another preferred example, the primer pair set further comprises a fourth primer pair, the fourth primer pair specifically amplifies an internal reference RNase P gene, and the fourth primer pair comprises:
a forward primer shown as SEQ ID NO. 10; and, a reverse primer as shown in SEQ ID NO. 11.
In a second aspect of the present invention, there is provided a probe set for detecting a novel coronavirus and an Omicron mutant strain thereof, the probe set comprising:
a first probe with a nucleotide sequence shown as SEQ ID NO. 9.
In another preferred embodiment, the probe set further comprises a second probe having a nucleotide sequence shown in SEQ ID NO. 3.
In another preferred embodiment, the probe set further comprises a third probe with the nucleotide sequence shown in SEQ ID NO. 6.
In another preferred embodiment, the probe set further comprises a fourth probe having a nucleotide sequence shown in SEQ ID NO. 12.
In another preferred embodiment, the 5' end of each probe comprises a fluorescent reporter group; and/or, the 3' end of each probe comprises a fluorescence quenching group.
In another preferred embodiment, the fluorescent reporter groups labeled by the probes are different from each other.
In a third aspect of the present invention, there is provided a kit for detecting a novel coronavirus and its omitron mutant strain, the kit comprising a primer set for detecting a novel coronavirus and its omitron mutant strain, the primer set comprising: a forward primer shown as SEQ ID NO. 7; and, a reverse primer as shown in SEQ ID NO. 8.
In another preferred embodiment, the kit further comprises a probe, wherein the probe specifically targets PCR amplification products of a forward primer shown by SEQ ID NO.7 and a reverse primer shown by SEQ ID NO. 8.
In another preferred embodiment, the kit comprises the primer pair set according to the first aspect of the present invention.
In another preferred embodiment, the kit further comprises a probe set according to the second aspect of the present invention.
In another preferred embodiment, the kit further comprises one or more components selected from the group consisting of: hot start Taq enzyme, reverse transcriptase, UDG enzyme (uracil-N-glycosylase), dNTPs, MgCl2。
In another preferred embodiment, the kit further comprises a negative quality control product.
In another preferred embodiment, the kit further comprises a positive quality control substance.
In another preferred embodiment, the kit includes a first container, and the first container contains a primer-probe mixture, the primer-probe mixture including: the nucleotide sequences shown in SEQ ID NO.1 to SEQ ID NO. 12.
In another preferred embodiment, the first container further comprises: MgCl2And dNTPs.
In another preferred example, the kit further comprises a second container, wherein the second container contains an enzyme mixture, and the enzyme mixture contains a hot start Taq enzyme, a reverse transcriptase, and a uracil glycosylase (UNG)).
In another preferred embodiment, the kit comprises a third container, and the third container contains negative quality control materials.
In another preferred embodiment, the kit comprises a fourth container, and the fourth container contains a positive quality control product.
In a fourth aspect of the present invention, there is provided a method for detecting a novel coronavirus and an Omicron mutant strain thereof, the method comprising the steps of:
(1) providing a sample of an object to be detected;
(2) preparing a fluorescent quantitative PCR reaction system and carrying out fluorescent quantitative PCR detection:
wherein, the fluorescent quantitative PCR reaction system comprises: the sample provided in step (1), the primer set of the first aspect of the invention, and the probe set of the second aspect of the invention.
In another preferred embodiment, the sample is a nucleic acid sample.
In another preferred example, the sample may be from a throat swab sample, an alveolar lavage sample, a blood sample, a sputum sample, a stool sample, or an environmental sample.
In another preferred embodiment, the method is a detection method for non-diagnostic purposes.
In another preferred embodiment, the fluorescent quantitative PCR reaction system further comprises a positive quality control substance and/or a negative quality control substance.
In another preferred embodiment, the PCR reaction system further comprises a PCR reaction enzyme system.
In a fifth aspect of the invention, there is provided the use of the primer set of the first aspect of the invention and/or the probe set of the second aspect of the invention for preparing a PCR detection kit for detecting novel coronaviruses and their omitron mutants.
In another preferred embodiment, the PCR is a fluorescent quantitative PCR.
It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.
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FIGS. 1 and 2 show the results of amplification of novel coronavirus and Omicron mutant strains detected using multiplex primer probe set 1 (including SEQ ID NO.1 to SEQ ID NO.12), respectively.
FIGS. 3 and 4 are amplification results for detection of novel coronavirus and Omicron mutant strains using multiplex primer probe set 2 (including SEQ ID NO.13 to SEQ ID NO.15, SEQ ID NO.28 to SEQ ID NO.30, SEQ ID NO.43 to SEQ ID NO.45, SEQ ID NO.52, SEQ ID NO.51, SEQ ID NO.42), respectively.
FIGS. 5 and 6 are amplification results for detection of novel coronavirus and Omicron mutant strains using multiplex primer probe set 3 (including SEQ ID NO.16 to 18, SEQ ID NO.31 to 33, SEQ ID NO.46 to 48, SEQ ID NO.24, SEQ ID NO.23, SEQ ID NO.12), respectively.
FIGS. 7 and 8 are amplification results for detection of novel coronavirus and Omicron mutant strains using multiplex primer probe set 4 (including SEQ ID NO.19 to 21, SEQ ID NO.34 to 36, SEQ ID NO.49, SEQ ID NO.50, SEQ ID NO.9, SEQ ID NO.10 to SEQ ID NO.12), respectively.
FIG. 9 shows the results of concentration gradient amplification of the target gene of ORF1ab of a novel coronavirus (2019-nCoV).
FIG. 10 shows the results of concentration gradient amplification of the N target gene of the novel coronavirus (2019-nCoV).
FIG. 11 shows the results of amplification of the S gene concentration gradient (wild type) of the novel coronavirus (2019-nCoV).
FIG. 12 shows the results of specific detection of pseudoviruses against human coronavirus (HKU 1).
FIG. 13 shows the result of detection of a negative sample (-);
FIG. 14 shows the results of detection of samples of Omicron variants;
FIG. 15 shows the results of the detection of a sample of the novel crown Delta variant.
Detailed Description
The invention develops a kit or a method for quickly detecting novel coronavirus and identifying the Omicron variant strain, and has very positive significance for epidemic prevention and control and treatment of patients with definite diagnosis. The novel coronavirus is qualitatively detected by simultaneously carrying out combined detection on three target genes of new crown ORF1ab and N, S in a 1 tube, and meanwhile, an S gene detection target region covers a specific mutation site of an Omicron variant, if the novel coronavirus is the Omicron variant, the S gene is lost without signals due to the mutation site, so that the Omicron variant and a non-Omicron variant can be further identified by judging whether the S gene is lost. The detection reagent provided by the invention can be used for detecting the novel coronavirus and the patient infected by the Omicron mutant strain thereof with high efficiency, high specificity, high stability and low cost.
Before the present invention is described, it is to be understood that this invention is not limited to the particular methodology and experimental conditions described, as such methodologies and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. As used herein, the term "about" when used in reference to a specifically recited value means that the value may vary by no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes 99 and 101 and all values in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).
Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now exemplified.
Real-time fluorescent PCR
Is a technique based on the principle of Fluorescence Resonance Energy Transfer (FRET). The real-time fluorescent PCR technology based on TaqMan fluorescent labeled probe utilizes thermostable DNA polymerase Taq enzyme to have polymerase activity in 5 '-3' direction and exonuclease activity in 5 '-3' direction of nucleotide sequence combined with target sequence in polymerization extension process. The TaqMan fluorescent probe is respectively marked with a fluorescence emitting group and a quenching group at the 5 ' end and the 3 ' end, and the 3 ' end of the probe is phosphorylated to prevent the probe from being extended in the PCR amplification process. The quencher inhibits fluorescent emission of the emissive moiety when the probe remains intact. Once the emitting group is separated from the quenching group, the inhibition is released, and the optical density at the emission wavelength of the fluorescent emitting group is increased and detected by the fluorescence detection system. The renaturation period probe is hybridized with template DNA, the Taq enzyme in the extension period moves along the DNA template along with the extension of the primer, when the Taq enzyme moves to the position of the probe, the 5' exonuclease activity of the Taq DNA polymerase can degrade the specific fluorescence labeled probe, the fluorescence reporter group is separated from the quenching group, and fluorescence is emitted.
Multiplex PCR
Multiplex PCR (multiplex PCR), also called multiplex PCR or multiplex PCR, is a PCR reaction in which two or more pairs of primers are added to the same PCR reaction system to simultaneously amplify multiple nucleic acid fragments, and the reaction principle, reaction reagents and operation process are the same as those of ordinary PCR.
There are many factors that affect multiplex PCR reactions, such as:
(1) the imbalance of the reaction system causes some dominant primers and templates thereof to be rapidly amplified in the previous rounds of reactions, and a large amount of amplification products are obtained, and the amplification products are good inhibitors of DNA polymerase. Therefore, the polymerization ability of polymerase is more and more strongly inhibited with the occurrence of a large amount of amplification products, and thus, primers and templates thereof which are at a disadvantage in the early stage are more difficult to react, and finally, the amount of amplification products is so small that they cannot be detected.
(2) The primer specificity, if the primer has stronger binding force with other non-target gene fragments in the system, the ability of the target gene to bind the primer is contended, thereby leading to the reduction of the amplification efficiency.
(3) The optimal annealing temperatures are different, a plurality of pairs of primers are placed in a system for amplification, and the optimal annealing temperatures of each pair of primers are required to be close to each other because the annealing temperatures for PCR reaction are the same.
(4) Primer dimers, including dimers between primers and hairpin structures formed by the primers themselves, are third-party DNA-mediated dimers, and these dimers, like non-specific primers, interfere with the competition between primers and target binding sites, affecting amplification efficiency.
Although several factors affecting amplification efficiency are mentioned above, more are not clear. To date, there is no effective method for clearly predicting amplification efficiency.
In a preferred embodiment of the present invention, the present invention provides a primer and probe combination for detecting a novel coronavirus, its Omicron mutant strain and reference gene, which has good specificity and high amplification efficiency and enables multiplex detection:
in another preferred embodiment of the present invention, the present invention provides a kit comprising the above primer-probe combination, the kit comprising:
an RT-PCR reaction solution comprising: MgCl2dNTPs, and the primer and probe combination of the invention;
an enzyme mixture comprising: hot start Taq enzyme, reverse transcriptase, uracil glycosylase (UNG));
negative quality control product: DEPC H2O；
Positive quality control product: contains a target gene of a novel coronavirus (2019-nCoV) ORF1ab, a target gene of a novel coronavirus (2019-nCoV) N, a specific mutation site of the novel coronavirus (2019-nCoV) Omicron and a target fragment plasmid of an internal reference gene.
In a preferred embodiment, the concentration of each primer is 0.3. mu.M.
In a preferred embodiment, each probe is present at a concentration of 0.15. mu.M.
The invention has the beneficial effects that:
(1) the invention establishes a detection method and a nucleic acid detection kit which can simultaneously detect novel coronavirus and an Omicron mutant strain thereof.
(2) The novel coronavirus and the Omicron mutant strain detection kit thereof have extremely high sensitivity.
(3) The detection method and the kit have extremely strong specificity aiming at the novel coronavirus Omicron mutant strain, and the detection result is stable and reliable.
(4) The invention designs and screens a large number of specific detection primers and probes for novel coronavirus and an Omicron mutant strain thereof to obtain a primer pair capable of specifically identifying mutation sites of the Omicron mutant strain, and through repeated verification, the primer pair is unexpectedly found to have no non-specific amplification on the wild novel coronavirus and other novel coronavirus mutant strains, so that the detection process of the novel coronavirus Omicron mutant strain is greatly simplified, and the accuracy for identifying the novel coronavirus Omicron mutant strain is obviously improved.
The invention is suitable for detecting novel coronavirus, provides reliable basis for virus identification, typing, prevention and control, and is worthy of popularization and application. In addition, the method of the present invention is also suitable for non-diagnostic purposes, for example, in the process of epidemic prevention and control, the detection method of the present invention is used for detecting the virus nucleic acid in the environment, and the virus nucleic acid information can be used as the requirement of public health management.
The present invention will be described in further detail with reference to the following examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures for conditions not specified in detail in the following examples are generally carried out under conventional conditions such as those described in molecular cloning, A laboratory Manual (Huang Petang et al, Beijing: scientific Press, 2002) by Sambrook. J, USA, or under conditions recommended by the manufacturer. Unless otherwise indicated, percentages and parts are by weight. The test materials and reagents used in the following examples are commercially available without specific reference. For example, the hot start Taq enzyme, UDG enzyme, and C-MMLV enzyme referred to in the examples of the present invention are available from Invitrogen corporation.
Example 1 kit for multiplex Rapid detection of novel coronavirus (2019-nCoV) and identification of Omicron mutant strains
In order to obtain a multiple primer probe system combination with good specificity and sensitivity, the inventor designs dozens of primers and probes in the research and development process, and combines the primers and the probes respectively to perform multiple tests.
Meanwhile, a human reference gene is designed to monitor the specimen collection, the nucleic acid extraction process and the PCR amplification process.
Finally, a set of primer and probe sequences with optimal sensitivity and specificity are determined.
As the content is excessive, partial results are selected and presented, partial primer probes are shown in the following table 1, and partial primer probe combination test results are shown in fig. 1, fig. 2, fig. 3 and fig. 4.
Experiments of the invention find that because the difference between the nucleic acid of the novel coronavirus Omicron mutant strain and the nucleic acid of the novel coronavirus wild type is very small, most primer pairs designed aiming at different mutation sites of the Omicron mutant strain can perform non-specific amplification on the wild type, and therefore, the Omicron mutant strain is difficult to accurately identify.
The invention designs and screens a large number of specific detection primers and probes aiming at novel coronavirus Omicron mutant strains to obtain primer pairs (SEQ ID NO.7 and SEQ ID NO. 8) capable of specifically identifying the corresponding mutation sites of the Omicron mutant strains, wherein the primer pairs specifically amplify nucleic acid fragments which are present in non-Omicron mutant strain nucleic acids and correspond to the Omicron mutant strain mutation sites, and when amplification products of the primer pairs exist in detection results, the fact that the Omicron mutant strain nucleic acids do not exist in samples is proved, and otherwise, the fact that the Omicron mutant strain nucleic acids exist in the samples is proved.
Repeated verification shows that the primer pair has no non-specific amplification to the Omicron mutant strain, so that the detection process of the novel coronavirus Omicron mutant strain is greatly simplified, and the accuracy of identifying the novel coronavirus Omicron mutant strain is obviously improved.
The multiplex primer probe combination 1 comprises SEQ ID NO. 1-SEQ ID NO. 12;
the multiple primer probe combination 2 comprises SEQ ID NO. 13-15, SEQ ID NO. 28-30, SEQ ID NO. 43-45, SEQ ID NO.52, SEQ ID NO.51 and SEQ ID NO. 42;
the multiplex primer probe combination 3 comprises SEQ ID NO. 16-18, SEQ ID NO. 31-33, SEQ ID NO. 46-48, SEQ ID NO.24, SEQ ID NO.23 and SEQ ID NO. 12;
the multiple primer probe combination 4 comprises SEQ ID NO. 19-21, SEQ ID NO. 34-36, SEQ ID NO. 49-50, SEQ ID NO.9, SEQ ID NO. 10-12;
the multiplex primer probe combination 5 comprises SEQ ID NO.22, SEQ ID NO.17, SEQ ID NO.18, SEQ ID NO.40, SEQ ID NO.41, SEQ ID NO.39, SEQ ID NO.43, SEQ ID NO.44, SEQ ID NO.45, SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO. 12;
the multiple primer probe combination 6 comprises SEQ ID NO. 25-27, SEQ ID NO. 4-6, SEQ ID NO. 7-9, SEQ ID NO.51, SEQ ID NO.42 and SEQ ID NO. 24.
The multiple detection results show that the amplification efficiency of the primer probe combination 1, the primer probe combination 5 and the primer probe combination 6 is high, and further performance tests can be carried out. When the primer probe combination 2, the primer probe combination 3 and the primer probe combination 4 are subjected to multiplex amplification, the amplification efficiency of partial targets is obviously inhibited, so that the sensitivity is poor, and nonspecific signals are generated for the Omicron variant.
FIGS. 1-8 show the results of detection of a portion of representative multiplex primer probe combinations.
FIGS. 1 and 2 show the results of amplification of novel coronavirus and Omicron mutant strains detected using multiplex primer probe set 1 (including SEQ ID NO.1 to SEQ ID NO.12), respectively. The concentration of the novel coronavirus wild type standard substance nucleic acid is 100 copies/mL, the wild type is detected by 100%, and the Omicron variant shows that S gene loss has no signal, so that the Omicron variant can be accurately identified.
FIGS. 3 and 4 are amplification results for detection of novel coronavirus and Omicron mutant strains using multiplex primer probe set 2 (including SEQ ID NO.13 to SEQ ID NO.15, SEQ ID NO.28 to SEQ ID NO.30, SEQ ID NO.43 to SEQ ID NO.45, SEQ ID NO.52, SEQ ID NO.51, SEQ ID NO.42), respectively. The concentration of the nucleic acid of the novel coronavirus wild-type standard substance is 100 copies/mL, the combination effect is shown as that the wild-type detection sensitivity is poor, and the Omicron variant strain still has a nonspecific signal after the S gene is lost, so that the Omicron variant strain cannot be accurately identified.
FIGS. 5 and 6 are amplification results for detection of novel coronavirus and Omicron mutant strains using multiplex primer probe set 3 (including SEQ ID NO.16 to 18, SEQ ID NO.31 to 33, SEQ ID NO.46 to 48, SEQ ID NO.24, SEQ ID NO.23, SEQ ID NO.12), respectively. The concentration of the nucleic acid of the novel coronavirus wild-type standard substance is 100 copies/mL, the combination effect is shown as that the wild-type detection sensitivity is poor, and the Omicron variant strain still has a nonspecific signal after the S gene is lost, so that the Omicron variant strain cannot be accurately identified.
FIGS. 7 and 8 are amplification results for detection of novel coronavirus and Omicron mutant strains using multiplex primer probe set 4 (including SEQ ID NO.19 to 21, SEQ ID NO.34 to 36, SEQ ID NO.49, SEQ ID NO.50, SEQ ID NO.9, SEQ ID NO.10 to SEQ ID NO.12), respectively. The concentration of the nucleic acid of the novel coronavirus wild-type standard substance is 100 copies/mL, the combination effect is shown as that the wild-type detection sensitivity is poor, and the Omicron variant strain still has a nonspecific signal after the S gene is lost, so that the Omicron variant strain cannot be accurately identified.
Based on the above-mentioned large amount of work, a novel coronavirus primer probe combination (including internal reference) with good specificity and high amplification efficiency is finally selected and selected as shown in table 2:
the present example provides a kit for multiplex rapid detection of novel coronavirus (2019-nCoV) and identification of Omicron mutant strain, comprising:
RT-PCR reaction (925. mu.l): MgCl2(4.5mM), dNTPs (0.2mM), a novel coronavirus (2019-nCoV) ORF1ab target gene, a novel coronavirus (2019-nCoV) N target gene and a novel coronavirus (2019-nCoV) Omicron specific mutation site, wherein the primer concentration is 0.3 mu M, and the probe concentration is 0.15 mu M.
Enzyme mix (75 μ L): including hot start Taq enzyme, reverse transcriptase, uracil glycosylase (UNG);
negative quality control product: DEPC H2O。
Positive quality control product: contains a target gene of a novel coronavirus (2019-nCoV) ORF1ab, a target gene of a novel coronavirus (2019-nCoV) N, a specific mutation site of the novel coronavirus (2019-nCoV) Omicron and a target fragment plasmid of an internal reference gene.
Example 2 method for multiplex Rapid detection of novel coronavirus (2019-nCoV) and identification of Omicron mutant strains
This example provides for the multiplex rapid detection of novel coronaviruses (2019-nCoV) and mutant strain Omicron identification using the kit of example 1.
Specimen type: oropharyngeal swab, nasopharyngeal swab.
Nucleic acid extraction:
a commercial RNA extraction kit, such as a nucleic acid extraction reagent based on a silica gel membrane centrifugal column method or a nucleic acid extraction reagent based on a magnetic bead method, is adopted, the operation is carried out according to the kit instruction, and finally 80 mu L of RNA solution is collected and directly detected.
Or stored at-80 ℃. The negative quality control product and the positive quality control product are extracted.
System configuration:
according to the total reaction number N (including the number of samples to be detected, negative quality control products and positive quality control products), 18.5 mul of RT-PCR amplification solution and 1.5 mul of enzyme mixed solution are added into each tube of PCR. Calculating the required total amount, mixing uniformly and then packaging into a special PCR reaction tube.
Sample adding:
adding a negative quality control product, a sample RNA solution and a positive quality control product into the PCR reaction tube which is filled with the reagents respectively, wherein the adding amount of the sample RNA solution and the positive quality control product is 10 mu L, tightly covering a tube cover, uniformly mixing, centrifuging, collecting the solution, and placing the solution at the bottom of the tube.
Performing on-machine amplification detection:
the fluorescence detection channel settings are shown in table 3:
the RT-PCR amplification program was set up as shown in Table 4:
6. and (4) analyzing results:
on the premise of satisfying the effective amplification, the judgment basis of the positive result of the target gene is shown in table 5:
the judgment criteria of the new crown Omicron variant are shown in the following table 6:
example 3 sensitivity detection
The wild-type novel coronavirus non-Omicron variant genomic RNA standard substance (standard substance No.: GBW (E) 091099) purchased from the national institute of measurement) was diluted to an appropriate concentration and then diluted 10-fold at 1X 10-fold ratios6copies/mL、1×105copies/mL、1×104copies/mL、1×103copies/mL、1×102copies/mL、1×101copies/mL。
The 6 gradient concentrations diluted with the novel coronavirus genomic RNA standard were tested using the test system and cycling parameters determined above. The amplification result Ct is shown in fig. 9 to 11.
The results showed that the sensitivity data was 1X 102copies/mL. The detection method is shown to have higher sensitivity, and the CT value of the detection result is shown in the following table 7.
Fig. 9 to 11: the 10-fold dilution concentrations of the novel coronavirus genome RNA standard substances in China metropolis are respectively 1 × 106copies/mL、1×105copies/mL、1×104copies/mL、1×103copies/mL、1×102copies/mL、1×101Amplification profiles of copies/mL. FIG. 9 shows the results of concentration gradient amplification of the target gene of ORF1ab of a novel coronavirus (2019-nCoV); FIG. 10 shows the results of concentration gradient amplification of the N target gene of the novel coronavirus (2019-nCoV); FIG. 11 shows the results of amplification of the S gene concentration gradient (wild type) of the novel coronavirus (2019-nCoV).
Example 4 specific assay
Pseudoviruses of endemic human coronavirus (HKU1, OC43, NL63 and 229E), SARS coronavirus, MERS coronavirus, influenza A H1N1 virus, influenza A H3N2 virus, influenza A H5N1 virus, influenza A H7N9 virus, influenza B virus, respiratory syncytial virus, parainfluenza virus, rhinovirus, adenovirus and enterovirus were diluted to 1X 106copies/mL was used as a specific test sample for detection.
The detection results show that the kit has good specificity, and the detection results show that 16 pathogens including human coronavirus (HKU1, OC43, NL63 and 229E), SARS coronavirus, MERS coronavirus, influenza A H1N1 virus, influenza A H3N2 virus, influenza A H5N1 virus, influenza A H7N9 virus, influenza B virus, respiratory syncytial virus, parainfluenza virus, rhinovirus, adenovirus and enterovirus are negative, and negative and positive quality control products are normally detected.
FIG. 12: pseudovirus-specific detection of human coronavirus (HKU 1).
Example 5 differential assay
In order to verify the accuracy and effectiveness of differential diagnosis of the Omicron detection kit and other variants of the new corona virus, pseudovirus samples of Alpha, Beta, Gamma, Delta, Lambda, Mu, Kappa and Iota of the new corona wild type and other variants are verified, the result of the Omicron detection kit shows that S genes of the Omicron variants are lost to be negative, S genes of the wild type and other variants are not lost to be positive, and the result shows that no cross reaction exists, so that the kit can accurately carry out differential diagnosis on the Omicron variants and non-Omicron variants. Partial detection results are shown in fig. 13 to 15:
FIG. 13 shows the result of detection of a negative sample (-);
FIG. 14 shows the results of detection of samples of Omicron variants;
FIG. 15 shows the results of the detection of a sample of the novel crown Delta variant.
All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.
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Claims (10)
1. A primer set for detecting a novel coronavirus and an Omicron variant thereof, wherein the primer set comprises a first primer pair:
the first primer pair comprises: a forward primer shown as SEQ ID NO. 7; and, a reverse primer as shown in SEQ ID NO. 8.
2. The primer set of claim 1, further comprising primer pairs for detecting a novel coronavirus ORF1ab gene, and/or an N gene.
3. The set of primer pairs of claim 1, further comprising one or more primer pairs selected from the group consisting of:
a second primer pair that specifically amplifies a novel coronavirus ORF1ab gene, the second primer pair comprising:
a forward primer shown as SEQ ID NO. 1; and, a reverse primer as shown in SEQ ID NO. 2;
a third primer pair that specifically amplifies a novel coronavirus N gene, the third primer pair comprising:
a forward primer shown as SEQ ID NO. 4; and, a reverse primer as shown in SEQ ID NO. 5;
a fourth primer pair for specifically amplifying an internal reference RNase P gene, the fourth primer pair comprising:
a forward primer shown as SEQ ID NO. 10; and, a reverse primer as shown in SEQ ID NO. 11.
4. A probe set for detecting a novel coronavirus and an Omicron mutant strain thereof, wherein the probe set comprises:
a first probe with a nucleotide sequence shown as SEQ ID NO. 9.
5. The probe set of claim 4, further comprising one or more probes selected from the group consisting of:
a second probe with a nucleotide sequence shown as SEQ ID NO. 3;
a third probe with a nucleotide sequence shown as SEQ ID NO. 6;
a fourth probe with the nucleotide sequence shown as SEQ ID NO. 12.
6. A kit for detecting a novel coronavirus and its Omicron mutant strain, comprising a primer set for detecting a novel coronavirus and its Omicron variant strain, said primer set comprising: a forward primer shown as SEQ ID NO. 7; and, a reverse primer as shown in SEQ ID NO. 8.
7. The kit of claim 6, wherein the kit comprises the primer set of any one of claims 1 to 3.
8. The kit of claim 7, further comprising the probe set of claim 4 or 5.
9. A method for the detection of novel coronaviruses and their omitron mutants for non-diagnostic purposes, characterized in that it comprises the steps of:
(1) providing a sample of an object to be detected;
(2) preparing a fluorescent quantitative PCR reaction system and carrying out fluorescent quantitative PCR detection:
wherein, the fluorescent quantitative PCR reaction system comprises: the sample provided in step (1), the set of primer pairs of claim 1, and the set of probes of claim 4.
10. Use of the primer set according to any one of claims 1 to 3, and/or the probe set according to any one of claims 4 to 5, for preparing a PCR detection kit for detecting novel coronaviruses and Omicron mutants thereof.
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Also Published As
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| CN116555497A (en) | 2023-08-08 |
| CN116555497B (en) | 2024-03-19 |
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