CN113817868A

CN113817868A - Primer, probe composition and kit for detecting novel coronavirus and variant thereof

Info

Publication number: CN113817868A
Application number: CN202110775408.6A
Authority: CN
Inventors: 刘亚宝; 韩英; 赵燕
Original assignee: Chengdu Fengji Biotechnology Co ltd
Current assignee: Chengdu Fengji Biotechnology Co ltd
Priority date: 2021-07-08
Filing date: 2021-07-08
Publication date: 2021-12-21
Anticipated expiration: 2041-07-08
Also published as: CN113817868B

Abstract

The invention discloses a primer, a probe composition and a kit for detecting novel coronavirus and variant thereof, which comprise specific primers and probes for ORF1ab gene, N gene and internal reference gene of SARS-CoV-2, and also comprise specific primers and probes for one or more mutation sites of S gene 69-70Del, L452R, E484K, E484Q and N501Y mutation sites, NSP6 gene 106/107/108Del mutation sites, E gene P71L mutation sites, ORF7A gene V82A mutation sites and ORF3A gene S253P mutation sites of SARS-CoV-2. The primer, the probe composition and the kit can detect mutation sites when detecting the novel coronavirus, and can distinguish one or more of a B.1.351 variant strain, a B.1.1.7 variant strain, a B.1.617 variant strain or a P.1 variant strain of the novel coronavirus; the reagent is a novel coronavirus and variant strain detection reagent which is rapid, low in cost and rich in variant information, and provides powerful help for global epidemic prevention and control and epidemic research.

Description

Primer, probe composition and kit for detecting novel coronavirus and variant thereof

Technical Field

The invention relates to the technical field of biological engineering, in particular to a primer, a probe composition and a kit for detecting novel coronavirus and variant thereof. More particularly relates to a kit for detecting nucleic acid of novel coronavirus and variant strains by adopting multiplex fluorescence PCR technology.

Background

At present, the mainstream method for detecting the novel coronavirus variant internationally is gene sequencing, but the gene sequencing consumes long time and is expensive and difficult to popularize in a large range. A novel coronavirus mutation site detection kit based on a QPCR (quantitative polymerase chain reaction) technical platform is provided by individual manufacturers such as Xiamen-derived organisms (Zeesan Biotech) and Huada genes (BGI Genomics), only one variant strain can be identified, a plurality of different variant strains cannot be identified simultaneously, and the clinical detection requirements cannot be well met.

Disclosure of Invention

The technical problem to be solved by the invention is to provide a primer, a probe composition and a kit for detecting a novel coronavirus and a variant thereof, so that a mutation site can be detected when the novel coronavirus is detected, one or more of a novel coronavirus B.1.351 variant, a novel coronavirus B.1.1.7 variant, a novel coronavirus B.1.617 variant or a novel coronavirus P.1 variant can be distinguished, and the existence of other types of variants is suggested.

In order to solve the technical problems, the invention adopts the following technical scheme:

a primer and probe composition for detecting novel coronavirus and variant thereof, which comprises a primer and a probe aiming at the specificity of ORF1ab gene, N gene and reference gene of SARS-CoV-2, and also comprises: primers and probes specific to one or more mutation sites of S gene 69-70Del, L452R, E484K, E484Q and N501Y mutation sites of SARS-CoV-2, NSP6 gene 106/107/108Del mutation sites, E gene P71L mutation sites, ORF7A gene V82A mutation sites, ORF3A gene S253P mutation sites; the primer and probe composition is used for detecting mutation sites when detecting novel coronavirus, and can distinguish one or more of novel coronavirus B.1.351 variant, B.1.1.7 variant, B.1.617 variant or P.1 variant.

The invention carries out the design of primers and probes by carrying out a large amount of screening, analysis and verification on 1,275,071 novel coronavirus gene sequences published on an outbreak data network and more than 10 typical variant strains and finally selecting the specific genes and mutation sites, can accurately identify various variant strains and well meet the requirements of clinical detection.

As a further improvement of the invention, a forward primer of a 69-70Del mutation site of the S gene is shown as SEQ ID NO.1, a reverse primer is shown as SEQ ID NO.2, and a probe is shown as SEQ ID NO. 3; the forward primer of the S gene L452R mutation site is shown as SEQ ID NO.4, the reverse primer is shown as SEQ ID NO.5, and the probe is shown as SEQ ID NO. 6; the forward primer of the S gene E484K mutation site is shown as SEQ ID NO.7, the reverse primer is shown as SEQ ID NO.10, and the probe is shown as SEQ ID NO. 11; the forward primer of the S gene E484Q mutation site is shown as SEQ ID NO.8, the reverse primer is shown as SEQ ID NO.10, and the probe is shown as SEQ ID NO. 11; the forward primer of the mutation site of the S gene N501Y is shown as SEQ ID NO.9, the reverse primer is shown as SEQ ID NO.10, and the probe is shown as SEQ ID NO. 11; the forward primer of the 106/107/108del mutation site of the NSP6 gene is shown as SEQ ID NO.12, the reverse primer is shown as SEQ ID NO.13, and the probe is shown as SEQ ID NO. 14; the forward primer of the E gene P71L mutation site is shown as SEQ ID NO.15, the reverse primer is shown as SEQ ID NO.16, and the probe is shown as SEQ ID NO. 17; the forward primer of the V82A mutation site of ORF7A gene is shown as SEQ ID NO.18, the reverse primer is shown as SEQ ID NO.19, and the probe is shown as SEQ ID NO. 20; the forward primer of the ORF3A gene S253P mutation site is shown as SEQ ID NO.21, the reverse primer is shown as SEQ ID NO.22, and the probe is shown as SEQ ID NO. 23.

The invention designs the primer and the probe aiming at the selected gene and the mutation site, considers a plurality of conventional design factors and design experience accumulation in the design process, and finally determines the specific probe and the primer aiming at the gene and the mutation site through clinical sample verification. The result accuracy of the primers and the probes for detecting the mutation sites is proved to be 100% by comparison with a commercial kit and clinical verification.

Further, as a preferred scheme, a forward primer of the ORF1ab gene is shown as SEQ ID NO.24, a reverse primer is shown as SEQ ID NO.25, and a probe is shown as SEQ ID NO. 26; and/or, the forward primer of the N gene is shown as SEQ ID NO.27, the reverse primer is shown as SEQ ID NO.28, and the probe is shown as SEQ ID NO. 29; and/or the internal reference gene is an ACTIN gene, a forward primer of the ACTIN gene is shown as SEQ ID NO.30, a reverse primer is shown as SEQ ID NO.31, and a probe is shown as SEQ ID NO. 32. Of course, it is understood that the primers and probes for the novel coronavirus may be other primers and probes known in the art, and the reference gene may be other conventional reference genes except the ACTIN gene.

Further, the same first fluorescent group is adopted at the 5' end of the probes of the S gene 69-70Del mutation site, the ORF1ab gene and the N gene; the same second fluorescent group is adopted at the 5' end of the probes of the S gene L452R, E484K, E484Q and N501Y mutation sites; the 5' end of the probe of the 106/107/108del mutation site of the NSP6 gene, the P71L mutation site of the E gene, the V82A mutation site of the ORF7A gene and the S253P mutation site of the ORF3A gene adopts the same third fluorescent group; and the 5' end of the probe of the internal reference gene adopts a fourth fluorescent group.

Furthermore, FAM fluorescent groups are adopted at the 5 'ends of probes of the S gene 69-70Del mutation sites, the ORF1ab gene and the N gene, and BHQ1 quenching groups are adopted at the 3' ends; the 5 'ends of the probes of the S genes L452R, E484K, E484Q and N501Y mutation sites all adopt ROX fluorescent groups, and the 3' ends all adopt BHQ2 quenching groups; the 5 'ends of probes of the 106/107/108del mutation site of the NSP6 gene, the P71L mutation site of the E gene, the V82A mutation site of the ORF7A gene and the S253P mutation site of the ORF3A gene all adopt HEX fluorescent groups, and the 3' ends all adopt BHQ1 quenching groups; the 5 'end of the probe of the internal reference gene adopts CY5 fluorescent group, and the 3' end adopts BHQ2 quenching group.

Furthermore, the primers and probes of the genes and the mutation sites are combined to form different mixed solutions; in one mixture, probes of ORF1ab gene and N gene exist simultaneously, and are different from the fluorescent group at the 5' end of the probe of other genes or mutation sites in the mixture; the 5' -end of each probe in each of the other mixed solutions has a different fluorophore; each mixture was used to perform multiplex fluorescent PCR reactions using one PCR tube, respectively.

The primers and the probes obtained by the design are mixed to form different mixed solution combinations for multiple fluorescence PCR detection, so that the PCR detection efficiency can be greatly improved.

Further, the mixed solution is 1 to 3.

Further, the mixed solution is PCR reaction solution A and PCR reaction solution B; the PCR reaction solution A contains primers and probes of ORF1ab gene, N gene, ORF3A gene S253P mutation site and S gene E484K mutation site; the PCR reaction solution B contains probes and genes of an S gene 69-70Del mutation site, an E gene P71L mutation site and an N501Y mutation site; the PCR reaction solution A and/or B contains primers and probes of reference genes; the composition is used for detecting novel coronavirus and novel coronavirus B.1.1.7 variant, B.1.351 variant or P.1 variant.

Further, the mixed solution is PCR reaction solution C and PCR reaction solution D; the PCR reaction solution C contains primers and probes of ORF1ab gene, N gene, ORF7A gene V82A mutation site, S gene E484Q mutation site and reference gene; the PCR reaction solution D contains primers and probes of an S gene 69/70Del mutation site, an E gene P71L mutation site, an S gene L452R mutation site and an ORF3A gene S253P mutation site; the composition is used for detecting novel coronavirus and novel coronavirus B.1.1.7 variant, B.1.351 variant, B.1.617 variant, B.1.617.1 variant, B.1.617.2 variant, B.1.617.3 variant or P.1 variant.

Further, the mixed solution is a PCR reaction solution E, PCR, a PCR reaction solution F and a PCR reaction solution G; the PCR reaction solution E contains primers and probes of ORF1ab gene, N gene, NSP6 gene 106/107/108Del mutation site and S gene E484K mutation site; the PCR reaction solution F contains primers and probes of ORF3A gene S253P mutation site, ORF7A gene V82A mutation site and S gene L452R mutation site; the PCR reaction solution G contains primers and probes of an S gene 69/70Del mutation site, an E gene P71L mutation site and an S gene E484Q mutation site; the PCR reaction solution E, F and/or G contains primers and probes of reference genes; the composition is used for detecting novel coronavirus and novel coronavirus B.1.1.7 variant, B.1.351 variant, B.1.617 variant or P.1 variant.

Further, a mixed solution used alone is a PCR reaction solution C; the PCR reaction solution C contains primers and probes of ORF1ab gene, N gene, ORF7A gene V82A mutation site, S gene E484Q mutation site and reference gene; the composition is used for detecting novel coronavirus B.1.617 variant, B.1.617.1 variant, B.1.617.2 variant or B.1.617.3 variant.

The invention also provides a kit for detecting the novel coronavirus and the variant thereof, wherein the kit comprises the primer and the probe composition for detecting the novel coronavirus and the variant thereof.

By adopting the technical scheme, the invention at least has the following advantages:

(1) the invention is based on multiplex fluorescence PCR amplification technology, detects ORF1ab gene, N gene, S gene 69-70Del, L452R, E484K, E484Q and N501Y mutation sites, NSP6 gene 106/107/108Del mutation site, E gene P71L mutation site, ORF7A gene V82A mutation site, ORF3A gene S253P mutation site and internal reference gene ACTIN of new coronavirus in a single sample, and can realize the following three purposes: (1) detecting a novel coronavirus; (2) identifying whether the strain is one of a novel coronavirus B.1.1.7 variant strain, a novel coronavirus B.1.351 variant strain, a novel coronavirus B.1.617 variant strain and a novel coronavirus P.1 variant strain; (3) suggesting the existence of other types of variants.

(2) The invention relates to a nucleic acid detection kit for realizing the identification of different novel coronavirus variant strains by adopting a QPCR (quantitative polymerase chain reaction) technical platform and through different combinations of primers/probes of specific mutation sites of the novel coronavirus variant strains. The invention can provide information whether the coronavirus variant strain is the virus variant strain or not while screening the novel coronavirus nucleic acid, is a novel coronavirus and variant strain detection method which are rapid, low in cost and rich in variant information, and provides a powerful tool for global epidemic situation prevention and control and epidemic disease research.

Drawings

The foregoing is only an overview of the technical solutions of the present invention, and in order to make the technical solutions of the present invention more clearly understood, the present invention is further described in detail below with reference to the accompanying drawings and the detailed description.

FIG. 1 is a diagram of the result of multiplex fluorescence PCR detection with primer and probe sets integrated, wherein: (a) is an amplification curve chart of ORF1ab gene/N gene; (b) an amplification curve chart of an internal reference gene ACTIN is shown; (c) comparison graphs of amplification curves of a wild type ORF3A gene and a mutant (S253P site mutation); (d) comparison of amplification curves for S gene wild type and mutant (E484K site mutation); (e) comparison of amplification curves of S gene wild type and mutant (69/70Del site mutation); (f) is a comparison graph of amplification curves of a wild type and a mutant (P71L site mutation) of the E gene; (g) comparison of amplification curves of S gene wild type and mutant (N501Y site mutation);

FIG. 2 shows the result of multiplex fluorescence PCR detection of the primer and probe combination II, wherein: (a) is an amplification curve chart of ORF1ab gene/N gene; (b) an amplification curve chart of an internal reference gene ACTIN is shown; (c) comparison of amplification curves for S gene wild type and mutant (E484Q site mutation); (d) comparison graphs of amplification curves of a wild type ORF7A gene and a mutant (V82A site mutation); (e) comparison of amplification curves of S gene wild type and mutant (69/70Del site mutation); (f) is a comparison graph of amplification curves of a wild type and a mutant (P71L site mutation) of the E gene; (g) comparison of amplification curves for S gene wild type and mutant (L452R site mutation); (h) is a comparison graph of amplification curves of a wild type ORF3A gene and a mutant (S253P site mutation).

FIG. 3 is a diagram showing the sequencing result of the mutation sites detected by the primer/probe set;

FIG. 4 is a diagram showing the sequencing results of the mutation sites detected by the primer/probe combination III.

Detailed Description

The technical scheme adopted by the invention is that specific primer/probe design is carried out aiming at ORF1ab gene, N gene, internal reference gene ACTIN, S gene 69-70Del, L452R, E484K, E484Q and N501Y mutant site, ORF1ab/NSP6 gene 106/107/108Del mutant site, E gene P71L mutant site, ORF7A gene V82A mutant site and ORF3A gene S253P mutant site of SARS-CoV-2, and multiple fluorescence PCR amplification can be realized in 1 to 3 PCR tubes by different combination modes of the primer/probe in a PCR reaction system, thereby completing the detection of novel coronavirus and variant thereof. The invention can detect mutation sites when detecting novel coronavirus for the first time, and can distinguish novel coronavirus B.1.351 variant, B.1.1.7 variant, B.1.617 variant or P.1 variant.

TABLE 1 primer and oligonucleotide probe sequences for detection of novel coronaviruses and variants thereof

The kit of the invention can be used for routine novel coronavirus nucleic acid screening. When clinical samples are aligned with a commercial Kit SARS-CoV-2 RT-PCR Kit (Sansure Biotech Inc.), the sensitivity of the reagent is more than 95 percent, the specificity is more than 98 percent, and the requirement of clinical detection is met.

The kit of the invention can also be used for detecting the novel coronavirus variant and judging whether the coronavirus variant is one of a B.1.351 variant, a B.1.1.7 variant, a B.1.617 variant or a P.1 variant. For other mutation detection results, the existence of other types of variant strains can be suggested, and further the mutation detection results can be identified through gene sequencing.

The invention verifies all samples with detected mutation sites by adopting a PCR + Sanger sequencing method, and the accuracy is 100%.

The following combination examples are provided to illustrate the present invention in detail.

Example 1

This example provides reagents for detecting coronavirus and variants thereof with various primer/probe combinations

1. Primer/probe set unification

1.1 PCR reaction solution contains: 2 XPCR reaction buffer solution, 0.1 to 0.5mmol/L deoxyribonucleoside triphosphate, 0.2 to 0.6 mu mol/L upstream primer and downstream primer of a target gene, 0.1 to 0.4 mu mol/L probe of the target gene, 0.1 to 0.3 mu mol/L upstream primer and downstream primer of an internal standard gene for amplification, and 0.05 to 0.2 mu mol/L probe of the internal standard.

1.2 PCR enzyme mixture containing: 1U/mul-5U/mul Taq DNA polymerase, 1U/mul-5U/mul reverse transcriptase and 1U/mul-10U/mul RNase inhibitor.

1.3 this combination can detect new coronavirus B.1.1.7 variant, B.1.351 variant or P.1 variant.

2. Primer/probe combination two

2.1 PCR reaction solution contains: 2 XPCR reaction buffer solution, 0.1 to 0.5mmol/L deoxyribonucleoside triphosphate, 0.2 to 0.6 mu mol/L upstream primer and downstream primer of a target gene, 0.1 to 0.4 mu mol/L probe of the target gene, 0.1 to 0.3 mu mol/L upstream primer and downstream primer of an internal standard gene for amplification, and 0.05 to 0.2 mu mol/L probe of the internal standard.

2.2 PCR enzyme mixture contains: 1U/mul-5U/mul Taq DNA polymerase, 1U/mul-5U/mul reverse transcriptase and 1U/mul-10U/mul RNase inhibitor.

2.3 this combination can detect the new coronavirus B.1.1.7 mutant, B.1.351 mutant, B.1.617 mutant, B.1.617.1 mutant, B.1.617.2 mutant, B.1.617.3 mutant or P.1 mutant.

3. Primer/probe combination III

3.1 PCR reaction solution contains: 2 XPCR reaction buffer solution, 0.1 to 0.5mmol/L deoxyribonucleoside triphosphate, 0.2 to 0.6 mu mol/L upstream primer and downstream primer of a target gene, 0.1 to 0.4 mu mol/L probe of the target gene, 0.1 to 0.3 mu mol/L upstream primer and downstream primer of an internal standard gene for amplification, and 0.05 to 0.2 mu mol/L probe of the internal standard.

3.2 PCR enzyme mixture contains: 1U/mul-5U/mul Taq DNA polymerase, 1U/mul-5U/mul reverse transcriptase and 1U/mul-10U/mul RNase inhibitor.

3.3 this combination can detect the new coronavirus B.1.1.7 variant, B.1.351 variant, B.1.617 variant or P.1 variant.

4. Primer/probe combination four

4.1 PCR reaction solution contains: 2 XPCR reaction buffer solution, 0.1 to 0.5mmol/L deoxyribonucleoside triphosphate, 0.2 to 0.6 mu mol/L upstream primer and downstream primer of a target gene, 0.1 to 0.4 mu mol/L probe of the target gene, 0.1 to 0.3 mu mol/L upstream primer and downstream primer of an internal standard gene for amplification, and 0.05 to 0.2 mu mol/L probe of the internal standard.

4.2 PCR enzyme mixture contains: 1U/mul-5U/mul Taq DNA polymerase, 1U/mul-5U/mul reverse transcriptase and 1U/mul-10U/mul RNase inhibitor.

4.3 this combination can detect new coronavirus B.1.617 variant, B.1.617.1 variant, B.1.617.2 variant or B.1.617.3 variant.

Example 2

This example provides a method for using a detection kit for detecting novel coronavirus and variants thereof

1. Collecting samples:

1.1 applicable samples: nasopharyngeal swab, oropharyngeal swab.

1.2 sample collection method: a sample collection is performed using a swab in the naso-pharynx or oro-pharynx of the patient, the swab is inserted to an appropriate depth, turned in situ for a few seconds to absorb the subject secretions, and the swab is removed with slow rotation. Immediately after the sample is collected, the swab is put into a virus sampling tube containing 1-3 ml of virus preservation solution.

1.3 the collected specimen should be sent for inspection in time, and the specimen should be stored at 4 ℃ within 24 hours.

1.4 the sample is transported by using an ice kettle or a foam box for adding ice and sealing.

2. Sample pretreatment:

2.1 nucleic acid extraction of samples

Any commercial Viral nucleic acid RNA extraction Kit (e.g., Qiagen Viral RNA Mini Kit, etc.) can be used, and the extraction procedure is performed according to the instructions of the commercial Kit. The purified nucleic acid can be used for subsequent PCR amplification. The specific operation steps of the Qiagen Viral RNA Mini Kit are as follows:

2.1.1 to a 1.5ml EP tube 560. mu.l of buffer AVL was added.

2.1.2 mu.l of the specimen (or quality control substance) was added to the aforementioned EP tube, vortexed for 15 seconds, and the virus was lysed at room temperature for 10 minutes.

2.1.3 the EP tube was subjected to instantaneous centrifugation to remove water droplets from the interior of the lid. Add 560. mu.l of absolute ethanol, vortex mix, and centrifuge instantaneously to remove water droplets in the lid.

2.1.4 carefully aspirate 630. mu.l of solution into a QIAamp mini column (2ml collection tube), cover it, and centrifuge at 8000rpm for 1 minute. The QIAamp mini column was placed in a clean 2ml collection tube, and the collection tube containing the filtrate was discarded.

2.1.5 carefully open QIAamp mini column, add 500. mu.l buffer AW 1. The lid was closed and centrifuged at 8000rpm for 1 minute. The QIAamp mini column was placed in a clean 2ml collection tube and the collection tube containing the filtrate was discarded.

2.1.6 carefully open QIAamp mini column, add 500. mu.l buffer AW 2. The lid was closed and centrifuged at 14000rpm for 3 minutes.

2.1.7 carefully open the QIAamp mini column, repeat step 2.1.6 once.

2.1.8 QIAamp mini-column was placed in a new 2ml collection tube and the old collection tube was discarded along with the filtrate. Centrifuge at 14000rpm for 1 minute.

2.1.9 QIAamp mini-columns were placed in clean 1.5ml EP tubes. The old collection tube containing the filtrate was discarded. Carefully open the lid and add 60 μ buffer AVE. The lid was closed and then allowed to stand at room temperature for 1 minute.

2.1.10 centrifugation at 8000rpm for 1 minute. The QIAamp mini column was discarded, and the EP tube containing the eluate was retained and subsequently available for PCR amplification.

2.2 sample processing for direct PCR (direct PCR)

The method of directly carrying out PCR after the sample is treated by adding the nucleic acid releasing agent can be adopted, the operation of purification equipment and a purification process is not needed, and the method is convenient and quick.

2.2.1 vortex to shake resuspend the sample.

2.2.2 in EP tube, 20. mu.L of viral nucleic acid releasing agent (Chengdifuji organisms) and 2. mu.L of RNA protecting agent were added and mixed well.

2.2.3 mu.L of sample (or quality control product) is added into the EP tube containing the nucleic acid releasing agent, is blown and mixed evenly, is kept stand for 10 minutes at room temperature, and can be used for PCR amplification subsequently.

3. Adding a reagent:

3.1 adding the reagent for detecting the novel coronavirus and the variant thereof with a plurality of different primer/probe combinations into a PCR tube. Wherein,

(1) adding 15 mu L of combined novel coronavirus and variant strain detection reagent thereof and 1.5 mu L of PCR enzyme mixed liquor into a PCR tube 1# and a PCR tube 2# respectively;

(2) adding 15 μ L of the detection reagent for the combined II coronavirus and the variant thereof and 1.5 μ L of PCR enzyme mixed solution into a PCR tube 1# and a PCR tube 2# respectively;

(3) adding 15 μ L of the third combined novel coronavirus and variant strain detection reagent thereof and 1.5 μ L of PCR enzyme mixed solution into a PCR tube 1#, a PCR tube 2# and a PCR tube 3# respectively;

(4) adding 15 μ L of the novel coronavirus and variant detection reagent thereof and 1.5 μ L of PCR enzyme mixed solution into the PCR tube 1# respectively;

3.2 the detection reagent of the four primer/probe combinations can be selected for subsequent detection. mu.L of the nucleic acid (2.1 or 2.2) of the treated sample (or quality control) was added to the PCR amplification system. And (4) covering the PCR tube cover, and waiting to be installed on the machine.

3.3 examples of the manner of applying the sample

3.3.1 sample application methods of combination one and combination two

Injecting NC-negative quality control; PC-Positive quality control

3.3.2 sample addition method of combination III

Injecting NC-negative quality control; PC-Positive quality control

3.3.3 sample application method of combination IV

Injecting NC-negative quality control; PC-Positive quality control

Setting and operating a PCR amplification program:

4.1 in a fluorescent quantitative PCR instrument such as ABI7500, Bio-Rad CFX96, Roche LightCycler 480,

PCR amplification was performed on SLAN-96S.

4.2 open the instrument and place the PCR tube to be tested.

4.3 set up the amplification program. The corresponding fluorescent channel FAM channel (Reporter: FAM, Quencher: None), HEX channel (Reporter: HEX, Quencher: None), ROX channel (Reporter: ROX, Quencher: None), CY5 channel (Reporter: CY5, Quencher: None) were selected. And carrying out sample editing.

4.4 run the amplification program.

4.5 specific amplification procedures were as follows:

5. and (4) analyzing results:

5.1 after the reaction is finished, the instrument automatically stores the result, the instrument can automatically analyze the result by using the software of the instrument (or manually adjust the starting value, the ending value and the threshold line value of the baseline to analyze the result), and then record the Ct value and the result of the sample. The intersection of the amplification curve with the threshold line, referred to as ct (cycle threshold); the instrument software can judge the detection result according to the Ct value of each sample.

5.2 for the sample with Ct value less than or equal to 40, reporting as Positive (POS); negative (NEG) was reported for samples assayed with no Ct value or Ct value > 40. If the Ct value of the internal standard is greater than 38 or is not shown, the detection result of the sample is invalid, the reason should be searched and eliminated, and the sample is subjected to repeated tests.

6. And (4) interpretation of results:

6.1 primer/Probe set integrated new type coronavirus and its variant detection reagent result interpretation

1) ORF1/N (FAM signal in tube A), S69/70 Del (FAM signal in tube B), and ACTIN (CY 5) were judged as NEG or POS as they were according to 5.2.

2) If the CT value (HEX signal for a tube) of S253P — the CT value (FAM signal for a tube) of ORF1 >6, or S253P has no CT value, S253P is NEG. Conversely, S253P is a POS.

3) If the CT value of E484K (ROX signal in a tube) -the CT value of ORF1 (FAM signal in a tube) >6, or E484K has no CT value, then E484K is NEG. In contrast, E484K is a POS.

4) If the CT value of P71L (HEX signal for B tube) -the CT value of ORF1 (FAM signal for a tube) >6, or P71L has no CT value, then P71L is NEG. Conversely, P71L is the POS.

5) If the CT value of N501Y (ROX signal for B tube) -the CT value of ORF1 (FAM signal for a tube) >6, or N501Y has no CT value, N501Y is NEG. In contrast, N501Y is POS.

6.2 primer/Probe combination two novel coronavirus and variant detection reagent result interpretation thereof

1) ORF1/N (FAM signal in tube A), S69/70 Del (FAM signal in tube B), and ACTIN (CY5 signal in tube A) were judged as NEG or POS as they were according to 5.2.

2) If the CT value (HEX signal in tube a) of V82A — CT value (FAM signal in tube a) of ORF1 >6, or V82A has no CT value, V82A is NEG. In contrast, V82A is the POS.

3) If the CT value of E484Q (ROX signal in a tube) -the CT value of ORF1 (FAM signal in a tube) >6, or E484Q has no CT value, then E484Q is NEG. In contrast, E484Q is a POS.

5) If the CT value of L452R (ROX signal in B-tube) -CT value of ORF1 (FAM signal in A-tube) >6, or L452R has no CT value, then L452R is NEG. In contrast, L452R is a POS.

6) If the CT value of S253P (CY5 signal for B tube) -the CT value of ORF1 (FAM signal for a tube) >6, or S253P has no CT value, S253P is NEG. Conversely, S253P is a POS.

6.3 primer/Probe combination III for determining results of reagents for detecting novel coronavirus and variant thereof

1) ORF1/N (FAM signal in tube A), NSP 6106-108 Del (HEX signal in tube A), S69/70 Del (FAM signal in tube C), and ACTIN (CY 5) were directly judged as NEG or POS at 5.2.

2) If the CT value of E484K (ROX signal in a tube) -the CT value of ORF1 (FAM signal in a tube) >6, or E484K has no CT value, then E484K is NEG. In contrast, E484K is a POS.

3) If the CT value (FAM signal for B tube) -the CT value of ORF1 (FAM signal for a tube) of S253P >6, or if there is no CT value of S253P, then NEG is determined at S253P. On the contrary, if S253P is POS.

4) If the CT value of V82A (HEX signal for B tube) -the CT value of ORF1 (FAM signal for a tube) >6, or V82A has no CT value, V82A is NEG. In contrast, V82A is the POS.

6) If the CT value of P71L (HEX signal for C tube) -the CT value of ORF1 (FAM signal for a tube) >6, or P71L has no CT value, then P71L is NEG. Conversely, P71L is the POS.

7) If the CT value of E484Q (ROX signal for B-tube) -CT value of ORF1 (FAM signal for A-tube) >6, or E484Q has no CT value, E484Q is NEG. In contrast, E484Q is a POS.

6.4 primer/Probe combination four novel coronavirus and variant strain detection reagent result interpretation thereof

1) ORF1/N (FAM signal) and ACTIN (CY5 signal) were judged as NEG or POS as they were according to 5.2.

2) V82A is NEG if the CT value (HEX signal) -the CT value (FAM signal) >6 of ORF1 of V82A, or if V82A has no CT value. In contrast, V82A is the POS.

3) E484Q is NEG if the CT value (ROX signal) -CT value FAM signal of ORF 1) >6 of E484Q, or E484Q has no CT value. In contrast, E484Q is a POS.

Example 3

This example provides the testing of reagents for detecting novel coronaviruses and variants thereof prepared by primer/probe set integration.

1. Sample preparation: the in vitro transcription product RNA1 of the DNA template (SEQ ID NO.33) contains RNA segments of novel coronavirus ORF1 gene, N gene, S gene E484K mutation site, S gene 69/70Del mutation site, S gene N501Y mutation site, ORF3A gene S253P mutation site and E gene P71L mutation site, and the in vitro transcription product RNA2 of the DNA template (SEQ ID NO.34) contains RNA segments of ORF3A gene, S gene and E gene.

2. The method comprises the following steps: according to the operation steps of the embodiment 2, the RNA fragments are diluted to 3pg/ul concentration, and different RNA fragments are parallelly detected on an ABI7500 instrument, and result analysis and statistics are carried out.

3. The results of the detection are shown in FIG. 1.

4. In conclusion, the reagent (combination one) of the invention can detect novel coronavirus ORF1 gene and N gene, and simultaneously detect S gene E484K mutation site, S gene 69/70Del mutation site, S gene N501Y mutation site, E gene P71L mutation site and ORF3A gene S253P mutation site of the novel coronavirus, thereby distinguishing corresponding mutant strains.

Example 4

This example provides a test for the detection of reagents for the novel coronavirus and variants thereof prepared from primer/probe combination two.

1. Sample preparation: RNA3, which is an in vitro transcription product of the DNA template (SEQ ID NO.35), contains RNA fragments of ORF1 gene, N gene, S gene E484Q mutation site, S gene HV69-70Del mutation site, S gene L452R mutation site, E gene P71L mutation site, ORF3A gene S253P mutation site, ORF7A gene V82A mutation site, and RNA4, which is an in vitro transcription product of the DNA template (SEQ ID NO.36), contains RNA fragments of ORF3A gene, ORF7A gene, S gene, E gene.

3. The results of the detection are shown in FIG. 2.

4. In conclusion, the reagent (combination II) of the invention, the detectable novel coronavirus ORF1 gene and the N gene, and simultaneously detect the novel coronavirus S gene E484Q mutation site, S gene 69-70Del mutation site, S gene L452R mutation site, E gene P71L mutation site, ORF3A gene S253P mutation site and ORF7A gene V82A mutation site, thereby distinguishing corresponding mutant strains.

Example 5

This example provides the clinical performance evaluation of novel coronavirus and variant kits thereof prepared by primer/probe set integration.

1. Sample source: 6 samples of the novel coronavirus which had been validated in the third party medical laboratory.

2. The detection method comprises the following steps: according to the method disclosed by the invention, the same sample is subjected to parallel detection on an ABI7500 instrument according to the operation steps of the embodiment 2, and the result analysis and statistics are carried out.

3. The detection result is specifically as follows:

verified by a third-party medical examination laboratory, the samples 1-4 in the embodiment are B.1.1.7 variant strains, the sample 5 is a negative sample, and the sample 6 is a common new crown sample, and the result is consistent with the interpretation result of the reagent.

4. And (3) mutation result analysis: the mutation sites detected by the reagent are analyzed by PCR and Sanger sequencing, and the accuracy of the mutation sites is 100%. The details are shown in the S HV69-70Del mutation site sequencing result and S N501Y mutation site sequencing result in FIG. 3.

5. In conclusion, the kit (combination one) can correctly detect the novel coronavirus and the variant thereof, and meets the clinical use requirement.

Example 6

This example provides the clinical performance evaluation of the novel coronavirus and variant kit thereof prepared from primer/probe combination III.

1. Sample source: 10 samples of the novel coronavirus that had been validated in a third party medical laboratory.

3. The detection result is specifically as follows:

verified by a third-party medical examination laboratory, the sample variant in the embodiment is consistent with the interpretation result of the reagent.

4. And (3) mutation result analysis: the mutation sites detected by the reagent are analyzed by PCR and Sanger sequencing, and the accuracy of the mutation sites is 100%. Specifically, see NSP 6106/107/108 Del mutation site sequencing results, S E484K mutation site sequencing results, ORF3A S253P mutation site sequencing results, ORF7A V82A mutation site sequencing results, S L452R mutation site sequencing results, S HV69-70Del mutation site sequencing results, E P71L mutation site sequencing results, S E484Q mutation site sequencing results in fig. 4.

5. In conclusion, the kit (combination III) can correctly detect the novel coronavirus and the variant thereof, and meets the clinical use requirement.

The above description is only for the purpose of illustrating preferred embodiments of the present invention and is not to be construed as limiting the present invention. Various modifications and alterations to this invention will become apparent to those skilled in the art. Any modification of the primer probe sequence, or change of the combination of the primer probe, or change of the fluorescent label of the probe, or use of the complementary sequence of the probe, etc. should be considered to be within the spirit and principle of the present invention. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present invention are included in the protection scope of the present invention.

Sequence listing

<110> Chengdu interpeak Biotechnology Co., Ltd

<120> primers, probe composition and kit for detecting novel coronavirus and variant thereof

<160> 36

<170> SIPOSequenceListing 1.0

<210> 1

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<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

gttccatgct atatctggga c 21

<210> 2

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

gcctcttatt atgttagact tctc 24

<210> 3

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

tactaagagg tttgataacc ctgtcctacc 30

<210> 4

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

agaggtgatg aagtcagac 19

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<212> DNA

<213> Artificial Sequence (Artificial Sequence)

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gtgattagac ttcctaaaca atctattcc 29

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<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

ccaccaacct tagaatcaag attgttagaa taccaag 37

<210> 7

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

ggtagcacag cttgtaatgg tgtca 25

<210> 8

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

ggtagcacag cttgtaatgg tgctc 25

<210> 9

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

tcatatggtt tccaacccac at 22

<210> 10

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

ccaaattagt agacttttta ggtcc 25

<210> 11

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

tctacatgca ccagcaactg tttgt 25

<210> 12

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

tggttgatac ttgtttgaag ct 22

<210> 13

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

gtgtcaagac attcataagt gtc 23

<210> 14

<211> 29

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

caccatcatc atacacagtt cttgctgtc 29

<210> 15

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

acaggtacgt taatagttaa tagcgt 26

<210> 16

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

gttcgtttag accagaagat cta 23

<210> 17

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

acactagcca tccttactgc gcttcg 26

<210> 18

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

gttacgtgcc agatcagc 18

<210> 19

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 19

gtctttcttt tgagtgtgaa gc 22

<210> 20

<211> 34

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 20

cttgaacttc ctcttgtctg atgaacagtt tagg 34

<210> 21

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 21

ttcacacaat cgacggttga c 21

<210> 22

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 22

tcgtactcat cagcttgtgc 20

<210> 23

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 23

cacgctagta gtcgtcgtcg gttcatc 27

<210> 24

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 24

ggggaacttc tcctgctaga at 22

<210> 25

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 25

cagacatttt gctctcaagc tg 22

<210> 26

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 26

ttgctgctgc ttgacagatt 20

<210> 27

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 27

ccctgtgggt tttacactta a 21

<210> 28

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 28

acgattgtgc atcagctga 19

<210> 29

<211> 28

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 29

ccgtctgcgg tatgtggaaa ggttatgg 28

<210> 30

<211> 17

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 30

cgagcgcggc tacagct 17

<210> 31

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 31

tccttaatgt cacgcacgat tt 22

<210> 32

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 32

accaccacgg ccgagcgg 18

<210> 33

<211> 1422

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 33

ggaagtctaa tctcaaacct tttgagagag atatttcaac tgaaatctat caggccggta 60

gcacaccttg taatggtgtt aaaggtttta attgttactt tcctttacaa tcatatggtt 120

tccaacccac ttatggtgtt ggttaccaac catacagagt agtagtactt tcttttgaac 180

ttctacatgc accagcaact gtttgtggac ctaaaaagtc tactaatttg gttaaaaaca 240

aatgtgtcaa tttcaacttc aatggtttaa caggcacagg tgttcttaca aatacctaca 300

acttgtgcta atgaccctgt gggttttaca cttaaaaaca cagtctgtac cgtctgcggt 360

atgtggaaag gttatggctg tagttgtgat caactccgcg aacccatgct tcagtcagct 420

gatgcacaat cgtttttaaa cgggtttgaa ttcaactcca ggcagcagta ggggaacttc 480

tcctgctaga atggctggca atggcggtga tgctgctctt gctttgctgc tgcttgacag 540

attgaaccag cttgagagca aaatgtctgg taaaggccaa caacaacaag gccaaacatg 600

tttgtttttc ttgttttatt gccactagtc tctagtcagt gtgttaatct tacaaccaga 660

actcaattac cccctgcata cactaattct ttcacacgtg gtgtttatta ccctgacaaa 720

gttttcagat cctcagtttt acattcaact caggacttgt tcttaccttt cttttccaat 780

gttacttggt tccatgctat atctgggacc aatggtacta agaggtttga taaccctgtc 840

ctaccattta atgatggtgt ttattttgct tccactgaga agtctaacat aataagaggc 900

tggatttttg gtactacttt agattcgaag acccagtccc tacttattgt taataacgct 960

actaatgttg ttattaaagt ctgtgaattt caatgtactc attcgtttcg gaagagacag 1020

gtacgttaat agttaatagc gtacttcttt ttcttgcttt cgtggtattc ttgctagtta 1080

cactagccat ccttactgcg cttcgattgt gtgcgtactg ctgcaatatt gttaacgtga 1140

gtcttgtaaa accttctttt tacgtttact ctcgtgttaa aaatctgaat tcttctagag 1200

ttcttgatct tctggtctaa acgaactaaa tattatatta gtttttctgt ttggaacttt 1260

aattttagga tgagcctgaa gaacatgtcc aaattcacac aatcgacggt tcacccggag 1320

ttgttaatcc agtaatggaa ccaatttatg atgaaccgac gacgactact agcgtgcctt 1380

tgtaagcaca agctgatgag tacgaactta tgtactcatt cg 1422

<210> 34

<211> 1428

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 34

ggaagtctaa tctcaaacct tttgagagag atatttcaac tgaaatctat caggccggta 60

gcacaccttg taatggtgtt gaaggtttta attgttactt tcctttacaa tcatatggtt 120

tccaacccac taatggtgtt ggttaccaac catacagagt agtagtactt tcttttgaac 180

ttctacatgc accagcaact gtttgtggac ctaaaaagtc tactaatttg gttaaaaaca 240

aatgtgtcaa tttcaacttc aatggtttaa caggcacagg tgttcttaca aatacctaca 300

acttgtgcta atgaccctgt gggttttaca cttaaaaaca cagtctgtac cgtctgcggt 360

atgtggaaag gttatggctg tagttgtgat caactccgcg aacccatgct tcagtcagct 420

gatgcacaat cgtttttaaa cgggtttgaa ttcaactcca ggcagcagta ggggaacttc 480

tcctgctaga atggctggca atggcggtga tgctgctctt gctttgctgc tgcttgacag 540

attgaaccag cttgagagca aaatgtctgg taaaggccaa caacaacaag gccaaacatg 600

tttgtttttc ttgttttatt gccactagtc tctagtcagt gtgttaatct tacaaccaga 660

actcaattac cccctgcata cactaattct ttcacacgtg gtgtttatta ccctgacaaa 720

gttttcagat cctcagtttt acattcaact caggacttgt tcttaccttt cttttccaat 780

gttacttggt tccatgctat acatgtctct gggaccaatg gtactaagag gtttgataac 840

cctgtcctac catttaatga tggtgtttat tttgcttcca ctgagaagtc taacataata 900

agaggctgga tttttggtac tactttagat tcgaagaccc agtccctact tattgttaat 960

aacgctacta atgttgttat taaagtctgt gaatttcaat gtactcattc gtttcggaag 1020

agacaggtac gttaatagtt aatagcgtac ttctttttct tgctttcgtg gtattcttgc 1080

tagttacact agccatcctt actgcgcttc gattgtgtgc gtactgctgc aatattgtta 1140

acgtgagtct tgtaaaacct tctttttacg tttactctcg tgttaaaaat ctgaattctt 1200

ctagagttcc tgatcttctg gtctaaacga actaaatatt atattagttt ttctgtttgg 1260

aactttaatt ttaggatgag cctgaagaac atgtccaaat tcacacaatc gacggttcat 1320

ccggagttgt taatccagta atggaaccaa tttatgatga accgacgacg actactagcg 1380

tgcctttgta agcacaagct gatgagtacg aacttatgta ctcattcg 1428

<210> 35

<211> 1532

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 35

gccggtagca caccttgtaa tggtgttcaa ggttttaatt gttactttcc tttacaatca 60

tatggtttcc aacccactaa tggtgttggt taccaaccat acagagtagt agtactttct 120

tttgaacttc tacatgcacc agcaactgtt tgtggaccta aaaagtctac taatttggtt 180

aaaaaccctg acggcgtaaa acacgtctat cagttacgtg ccagatcagc ttcacctaaa 240

ctgttcatca gacaagagga agttcaagaa ctttactctc caatttttct tattgttgcg 300

gcaatagtgt ttataacact ttgcttcaca ctcaaaagaa agacagaatg attgaacttt 360

ccaaatacct acaacttgtg ctaatgaccc tgtgggtttt acacttaaaa acacagtctg 420

taccgtctgc ggtatgtgga aaggttatgg ctgtagttgt gatcaactcc gcgaacccat 480

gcttcagtca gctgatgcac aatcgttttt aaacgggttt gaattcaact ccaggcagca 540

gtaggggaac ttctcctgct agaatggctg gcaatggcgg tgatgctgct cttgctttgc 600

tgctgcttga cagattgaac cagcttgaga gcaaaatgtc tggtaaaggc caacaacaac 660

aaggccaaac ctcaggactt gttcttacct ttcttttcca atgttacttg gttccatgct 720

atatctggga ccaatggtac taagaggttt gataaccctg tcctaccatt taatgatggt 780

gtttattttg cttccactga gaagtctaac ataataagag gctggatttt tggtactact 840

ttagattcga agacccagtc cctacttatt gttaataacg ctactaatgt tgttattaaa 900

gtctgtgaat ttcaatgtac tcattcgttt cggaagagac aggtacgtta atagttaata 960

gcgtacttct ttttcttgct ttcgtggtat tcttgctagt tacactagcc atccttactg 1020

cgcttcgatt gtgtgcgtac tgctgcaata ttgttaacgt gagtcttgta aaaccttctt 1080

tttacgttta ctctcgtgtt aaaaatctga attcttctag agttcttgat cttctggtct 1140

aaacgaacta aatattatat tagtttttct gtttggaact ttaattttag gtaattagag 1200

gtgatgaagt cagacaaatc gctccagggc aaactggaaa gattgctgat tataattata 1260

aattaccaga tgattttaca ggctgcgtta tagcttggaa ttctaacaat cttgattcta 1320

aggttggtgg taattataat taccggtata gattgtttag gaagtctaat ctcaaaccga 1380

tgagcctgaa gaacatgtcc aaattcacac aatcgacggt tcacccggag ttgttaatcc 1440

agtaatggaa ccaatttatg atgaaccgac gacgactact agcgtgcctt tgtaagcaca 1500

agctgatgag tacgaactta tgtactcatt cg 1532

<210> 36

<211> 1538

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 36

gccggtagca caccttgtaa tggtgttgaa ggttttaatt gttactttcc tttacaatca 60

tatggtttcc aacccactaa tggtgttggt taccaaccat acagagtagt agtactttct 120

tttgaacttc tacatgcacc agcaactgtt tgtggaccta aaaagtctac taatttggtt 180

aaaaaccctg acggcgtaaa acacgtctat cagttacgtg ccagatcagt ttcacctaaa 240

ctgttcatca gacaagagga agttcaagaa ctttactctc caatttttct tattgttgcg 300

gcaatagtgt ttataacact ttgcttcaca ctcaaaagaa agacagaatg attgaacttt 360

ccaaatacct acaacttgtg ctaatgaccc tgtgggtttt acacttaaaa acacagtctg 420

taccgtctgc ggtatgtgga aaggttatgg ctgtagttgt gatcaactcc gcgaacccat 480

gcttcagtca gctgatgcac aatcgttttt aaacgggttt gaattcaact ccaggcagca 540

gtaggggaac ttctcctgct agaatggctg gcaatggcgg tgatgctgct cttgctttgc 600

tgctgcttga cagattgaac cagcttgaga gcaaaatgtc tggtaaaggc caacaacaac 660

aaggccaaac ctcaggactt gttcttacct ttcttttcca atgttacttg gttccatgct 720

atacatgtct ctgggaccaa tggtactaag aggtttgata accctgtcct accatttaat 780

gatggtgttt attttgcttc cactgagaag tctaacataa taagaggctg gatttttggt 840

actactttag attcgaagac ccagtcccta cttattgtta ataacgctac taatgttgtt 900

attaaagtct gtgaatttca atgtactcat tcgtttcgga agagacaggt acgttaatag 960

ttaatagcgt acttcttttt cttgctttcg tggtattctt gctagttaca ctagccatcc 1020

ttactgcgct tcgattgtgt gcgtactgct gcaatattgt taacgtgagt cttgtaaaac 1080

cttcttttta cgtttactct cgtgttaaaa atctgaattc ttctagagtt cctgatcttc 1140

tggtctaaac gaactaaata ttatattagt ttttctgttt ggaactttaa ttttaggtaa 1200

ttagaggtga tgaagtcaga caaatcgctc cagggcaaac tggaaagatt gctgattata 1260

attataaatt accagatgat tttacaggct gcgttatagc ttggaattct aacaatcttg 1320

attctaaggt tggtggtaat tataattacc tgtatagatt gtttaggaag tctaatctca 1380

aaccgatgag cctgaagaac atgtccaaat tcacacaatc gacggttcat ccggagttgt 1440

taatccagta atggaaccaa tttatgatga accgacgacg actactagcg tgcctttgta 1500

agcacaagct gatgagtacg aacttatgta ctcattcg 1538

Claims

1. A primer and probe composition for detecting novel coronavirus and variant thereof, which comprises a primer and a probe aiming at the specificity of ORF1ab gene, N gene and reference gene of SARS-CoV-2, and is characterized by also comprising:

primers and probes specific to one or more mutation sites of S gene 69-70Del, L452R, E484K, E484Q and N501Y mutation sites of SARS-CoV-2, NSP6 gene 106/107/108Del mutation sites, E gene P71L mutation sites, ORF7A gene V82A mutation sites, ORF3A gene S253P mutation sites;

the primer and probe composition is used for detecting mutation sites when detecting novel coronavirus, and can distinguish one or more of novel coronavirus B.1.351 variant, B.1.1.7 variant, B.1.617 variant or P.1 variant.

2. The primer and probe composition for detecting novel coronavirus and variant thereof according to claim 1, wherein:

the forward primer of the S gene 69-70Del mutation site is shown as SEQ ID NO.1, the reverse primer is shown as SEQ ID NO.2, and the probe is shown as SEQ ID NO. 3;

the forward primer of the S gene L452R mutation site is shown as SEQ ID NO.4, the reverse primer is shown as SEQ ID NO.5, and the probe is shown as SEQ ID NO. 6;

the forward primer of the S gene E484K mutation site is shown as SEQ ID NO.7, the reverse primer is shown as SEQ ID NO.10, and the probe is shown as SEQ ID NO. 11;

the forward primer of the S gene E484Q mutation site is shown as SEQ ID NO.8, the reverse primer is shown as SEQ ID NO.10, and the probe is shown as SEQ ID NO. 11;

the forward primer of the mutation site of the S gene N501Y is shown as SEQ ID NO.9, the reverse primer is shown as SEQ ID NO.10, and the probe is shown as SEQ ID NO. 11;

the forward primer of the 106/107/108del mutation site of the NSP6 gene is shown as SEQ ID NO.12, the reverse primer is shown as SEQ ID NO.13, and the probe is shown as SEQ ID NO. 14;

the forward primer of the E gene P71L mutation site is shown as SEQ ID NO.15, the reverse primer is shown as SEQ ID NO.16, and the probe is shown as SEQ ID NO. 17;

the forward primer of the V82A mutation site of ORF7A gene is shown as SEQ ID NO.18, the reverse primer is shown as SEQ ID NO.19, and the probe is shown as SEQ ID NO. 20;

the forward primer of the ORF3A gene S253P mutation site is shown as SEQ ID NO.21, the reverse primer is shown as SEQ ID NO.22, and the probe is shown as SEQ ID NO. 23.

3. The primer and probe composition for detecting novel coronavirus and variant strain thereof according to claim 1 or 2, wherein:

the forward primer of the ORF1ab gene is shown as SEQ ID NO.24, the reverse primer is shown as SEQ ID NO.25, and the probe is shown as SEQ ID NO. 26;

and/or, the forward primer of the N gene is shown as SEQ ID NO.27, the reverse primer is shown as SEQ ID NO.28, and the probe is shown as SEQ ID NO. 29;

and/or the internal reference gene is an ACTIN gene, a forward primer of the ACTIN gene is shown as SEQ ID NO.30, a reverse primer is shown as SEQ ID NO.31, and a probe is shown as SEQ ID NO. 32.

4. The primer and probe composition for detecting novel coronavirus and variant strain thereof according to any one of claims 1 to 3, wherein:

the 5' ends of the probes of the S gene 69-70Del mutation site, the ORF1ab gene and the N gene adopt the same first fluorescent group;

the same second fluorescent group is adopted at the 5' end of the probes of the S gene L452R, E484K, E484Q and N501Y mutation sites;

the 5' end of the probe of the 106/107/108del mutation site of the NSP6 gene, the P71L mutation site of the E gene, the V82A mutation site of the ORF7A gene and the S253P mutation site of the ORF3A gene adopts the same third fluorescent group;

and the 5' end of the probe of the internal reference gene adopts a fourth fluorescent group.

5. The primer and probe composition for detecting novel coronavirus and variant thereof according to claim 4, wherein:

the 5 'ends of the probes of the S gene 69-70Del mutation site, the ORF1ab gene and the N gene all adopt FAM fluorescent groups, and the 3' ends all adopt BHQ1 quenching groups;

the 5 'ends of the probes of the S genes L452R, E484K, E484Q and N501Y mutation sites all adopt ROX fluorescent groups, and the 3' ends all adopt BHQ2 quenching groups;

the 5 'ends of probes of the 106/107/108del mutation site of the NSP6 gene, the P71L mutation site of the E gene, the V82A mutation site of the ORF7A gene and the S253P mutation site of the ORF3A gene all adopt HEX fluorescent groups, and the 3' ends all adopt BHQ1 quenching groups;

the 5 'end of the probe of the internal reference gene adopts CY5 fluorescent group, and the 3' end adopts BHQ2 quenching group.

6. The primer-probe combination for detecting novel coronavirus and variants thereof according to any one of claims 1 to 5, wherein the primer and probe combination of each gene and mutation site form different mixed solutions;

in one mixture, probes of ORF1ab gene and N gene exist simultaneously, and are different from the fluorescent group at the 5' end of the probe of other genes or mutation sites in the mixture;

the 5' -end of each probe in each of the other mixed solutions has a different fluorophore;

each mixture was used to perform multiplex fluorescent PCR reactions using one PCR tube, respectively.

7. The primer-probe composition for detecting novel coronavirus and variant thereof according to claim 6, wherein the mixture comprises 1 to 3 kinds of primers.

8. The primer-probe composition for detecting a novel coronavirus and a variant thereof according to claim 6, wherein the mixture is PCR reaction solution A and PCR reaction solution B;

the PCR reaction solution A contains primers and probes of ORF1ab gene, N gene, ORF3A gene S253P mutation site and S gene E484K mutation site;

the PCR reaction solution B contains probes and genes of an S gene 69-70Del mutation site, an E gene P71L mutation site and an N501Y mutation site;

the PCR reaction solution A and/or B contains primers and probes of reference genes;

the composition is used for detecting novel coronavirus and novel coronavirus B.1.1.7 variant, B.1.351 variant or P.1 variant.

9. The primer-probe composition for detecting a novel coronavirus and a variant thereof according to claim 6, wherein the mixture is PCR reaction solution C and PCR reaction solution D;

the PCR reaction solution C contains primers and probes of ORF1ab gene, N gene, ORF7A gene V82A mutation site, S gene E484Q mutation site and reference gene;

the PCR reaction solution D contains primers and probes of an S gene 69/70Del mutation site, an E gene P71L mutation site, an S gene L452R mutation site and an ORF3A gene S253P mutation site;

the composition is used for detecting novel coronavirus and novel coronavirus B.1.1.7 variant, B.1.351 variant, B.1.617 variant, B.1.617.1 variant, B.1.617.2 variant, B.1.617.3 variant or P.1 variant.

10. The primer-probe composition for detecting a novel coronavirus and a variant thereof according to claim 6, wherein the mixture is PCR reaction solution E, PCR, PCR reaction solution F and PCR reaction solution G;

the PCR reaction solution E contains primers and probes of ORF1ab gene, N gene, NSP6 gene 106/107/108Del mutation site and S gene E484K mutation site;

the PCR reaction solution F contains primers and probes of ORF3A gene S253P mutation site, ORF7A gene V82A mutation site and S gene L452R mutation site;

the PCR reaction solution G contains primers and probes of an S gene 69/70Del mutation site, an E gene P71L mutation site and an S gene E484Q mutation site;

the PCR reaction solution E, F and/or G contains primers and probes of reference genes;

the composition is used for detecting novel coronavirus and novel coronavirus B.1.1.7 variant, B.1.351 variant, B.1.617 variant or P.1 variant.

11. The primer-probe composition for detecting a novel coronavirus and a variant thereof according to claim 6, wherein the mixture is a PCR reaction solution C;

the composition is used for detecting novel coronavirus B.1.617 variant, B.1.617.1 variant, B.1.617.2 variant or B.1.617.3 variant.

12. A kit for detecting a novel coronavirus and a variant thereof, wherein the kit comprises the primer and probe composition for detecting a novel coronavirus and a variant thereof according to any one of claims 1 to 11.