CN113817868B

CN113817868B - Primer, probe composition and kit for detecting novel coronavirus and variant strain thereof

Info

Publication number: CN113817868B
Application number: CN202110775408.6A
Authority: CN
Inventors: 刘亚宝; 韩英; 宋雨城; 赵艳
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: 2023-08-18
Anticipated expiration: 2041-07-08
Also published as: CN113817868A

Abstract

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

Description

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

Technical Field

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

Background

At present, the mainstream method for detecting the novel coronavirus variant strain is gene sequencing, but the cost is high due to long time consumption of gene sequencing, and the method is difficult to popularize on a large scale. Individual manufacturers such as Xiamen-induced organisms (Zeesan Biotech) and Hua big genes (BGI Genomics) push out a novel coronavirus mutation site detection kit based on a QPCR technology platform, only one variant strain can be identified, a plurality of different variant strains cannot be identified at the same time, and the clinical detection requirement cannot be well met.

Disclosure of Invention

The invention aims to provide a primer, a probe composition and a kit for detecting novel coronaviruses and variants thereof, which can detect mutation sites when detecting novel coronaviruses, and can distinguish one or more of novel coronavirus variants B.1.351, B.1.1.7, B.1.617 or P.1 variants and indicate the existence of other types of variants.

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

a primer and probe composition for detecting novel coronaviruses and variants thereof, comprising primers and probes specific to the ORF1ab gene, N gene and internal reference gene of SARS-CoV-2, further comprising: specific primers and probes for one or more of the 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 of SARS-CoV-2; the primer and the probe composition are used for detecting mutation sites when detecting the novel coronavirus, and can distinguish one or more of the novel coronavirus B.1.351 variant, the novel coronavirus B.1.1.7 variant, the novel coronavirus B.1.617 variant or the novel coronavirus P.1 variant.

According to the invention, the primers and probes are designed by screening, analyzing and verifying a large number of 1,275,071 novel coronavirus gene sequences and more than 10 typical variants published on the outbreak data network, and finally selecting the specific genes and mutation sites, so that a plurality of different variants can be accurately identified, and the clinical detection requirements can be well met.

As a further improvement of the invention, 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 S gene N501Y mutation site 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 NSP6 gene 106/107/108del mutation site 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 mutation site of the E gene P71L 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 the 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 mutation site of the ORF3A gene S253P 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 genes and mutation sites, considers various conventional design factors and accumulation of design experience in the design process, and finally determines the specific probe and the primer aiming at the genes and the mutation sites through clinical sample verification. Compared with commercial kits and clinical verification prove that the accuracy of the results of detecting mutation sites by the primers and the probes is 100%.

Further, as a preferable scheme, 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 reference gene is an ACTIN gene, the forward primer of the ACTIN gene is shown as SEQ ID NO.30, the reverse primer is shown as SEQ ID NO.31, and the probe is shown as SEQ ID NO. 32. Of course, it will be understood that the primers and probes of the novel coronaviruses may be other primers and probes in the prior art, and that the reference gene may be any other conventional reference gene other than the ACTIN gene.

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

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

Further, the primers and probes of the genes and mutation sites are combined to form different mixed solutions; in one mixed solution, probes of the ORF1ab gene and the N gene exist at the same time and are different from fluorescent groups at the 5' end of probes of other genes or mutation sites in the mixed solution; the fluorescent groups at the 5' end of each probe in each other mixed solution are different; each mixture was used to perform multiple fluorescent PCR reactions using one PCR tube.

The primer and the probe obtained by the design are mixed to form different mixed liquid combinations for multiplex fluorescence PCR detection, so that the PCR detection efficiency can be greatly improved.

Further, the number of the mixed liquids is 1-3.

Further, the mixed solution is a PCR reaction solution A and a 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 liquid B contains an S gene 69-70Del mutation site, an E gene P71L mutation site, an N501Y mutation site and a probe; the PCR reaction liquid A and/or B contains primers and probes of internal reference genes; the composition is used for detecting novel coronaviruses and novel coronavirus B.1.1.7 variants, B.1.351 variants or P.1 variants.

Further, the mixed solution is a PCR reaction solution C and a 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 internal 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 coronaviruses, and novel coronavirus B.1.1.7 variants, B.1.351 variants, B.1.617 variants, B.1.617.1 variants, B.1.617.2 variants, B.1.617.3 variants or P.1 variants.

Further, the mixed solution is a PCR reaction solution E, 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 an ORF3A gene S253P mutation site, an ORF7A gene V82A mutation site and an 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; primer and probe of internal reference gene are contained in the PCR reaction liquid E, F and/or G; the composition is used for detecting novel coronaviruses and novel coronavirus B.1.1.7 variants, B.1.351 variants, B.1.617 variants or P.1 variants.

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 internal 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 strain thereof, and the kit comprises the primer and the probe composition for detecting the novel coronavirus and the variant strain thereof.

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

(1) The invention is based on multiplex fluorescence PCR amplification technology, and detects ORF1ab gene, N gene, S gene 69-70Del, L452R, E484K, E484Q and N501Y mutation site, 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 novel coronavirus in 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 B.1.351 variant strain, a B.1.617 variant strain and a P.1 variant strain; (3) suggesting the presence of other types of variants.

(2) The invention discloses a nucleic acid detection kit for identifying different novel coronavirus variants by adopting a QPCR technology platform and different combinations of primers/probes of specific mutation sites of the novel coronavirus variants. The invention can screen the novel coronavirus nucleic acid and provide information about whether the coronavirus is a virus variant, is a novel coronavirus and variant detection method with high speed, low cost and rich variant information, and provides a powerful tool for global epidemic prevention and control and epidemic disease research.

Drawings

The foregoing is merely an overview of the present invention, and the present invention is further described in detail below with reference to the accompanying drawings and detailed description.

FIG. 1 is a diagram showing the results of multiplex fluorescence PCR detection with primer and probe combinations, wherein: (a) is an amplification plot of ORF1ab gene/N gene; (b) is an amplification curve of reference gene ACTIN; (c) Comparison of amplification curves for wild type and mutant (S253P site mutation) of ORF3A gene; (d) Comparison of amplification curves for wild type and mutant of S gene (E484K site mutation); (e) Comparison of amplification curves for wild type and mutant (69/70 Del site mutation) of the S gene; (f) Comparison of amplification curves for wild type and mutant of E gene (P71L site mutation); (g) Comparison of amplification curves for wild type and mutant of S gene (N501Y site mutation);

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

FIG. 3 is a graph showing the sequencing result of a mutation site detected by a primer/probe combination;

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

Detailed Description

The technical scheme adopted by the invention is to design specific primers/probes aiming at ORF1ab gene, N gene, internal reference gene ACTIN of SARS-CoV-2, S gene 69-70Del, L452R, E484K, E Q and N501Y mutation site, 106/107/108Del mutation site of ORF1ab/NSP6 gene, P71L mutation site of E gene, V82A mutation site of ORF7A gene and S253P mutation site of ORF3A gene, and simultaneously realize multiplex fluorescence PCR amplification in 1-3 PCR tubes by different combination modes of the primers/probes in a PCR reaction system, thereby completing the detection of novel coronaviruses and variants thereof. The invention detects mutation sites when detecting novel coronaviruses 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 primers and oligonucleotide probe sequences for detection of novel coronaviruses and variants thereof

The kit can be used for screening conventional novel coronavirus nucleic acid. When compared with a commercial Kit SARS-CoV-2RT-PCR Kit (Sansure Biotech Inc.), the reagent of the invention has sensitivity of more than 95% and specificity of more than 98%, and meets the requirement of clinical detection.

The kit of the invention can also be used for detecting novel coronavirus variants and judging whether the coronavirus variants are one of B.1.351 variants, B.1.1.7 variants, B.1.617 variants or P.1 variants. For other mutation detection results, the presence of other types of variants can be suggested, which can be further identified by gene sequencing.

The invention adopts PCR+Sanger sequencing method to verify all samples with mutation sites, and the accuracy is 100%.

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

Example 1

This example provides novel coronavirus and variant detection reagents for a variety of different primer/probe combinations

1. Primer/probe combination one

1.1 The PCR reaction solution comprises: 2 XPCR reaction buffer, 0.1 mmol/L-0.5 mmol/L deoxyribonucleoside triphosphate, 0.2. Mu. Mol/L-0.6. Mu. Mol/L upstream primer and 0.1. Mu. Mol/L downstream primer of target gene, 0.1. Mu. Mol/L-0.4. Mu. Mol/L probe of target gene, 0.1. Mu. Mol/L upstream primer and 0.3. Mu. Mol/L downstream primer of internal standard gene for amplification, 0.05. Mu. Mol/L-0.2. Mu. Mol/L probe of internal standard.

1.2 The PCR enzyme cocktail comprises: 1U/. Mu.l to 5U/. Mu.l Taq DNA polymerase, 1U/. Mu.l to 5U/. Mu.l reverse transcriptase, 1U/. Mu.l to 10U/. Mu.l RNase inhibitor.

1.3 the combination allows the detection of novel coronavirus variants B.1.1.7, B.1.351 or P.1.

2. Primer/probe combination II

2.1 The PCR reaction solution comprises: 2 XPCR reaction buffer, 0.1 mmol/L-0.5 mmol/L deoxyribonucleoside triphosphate, 0.2. Mu. Mol/L-0.6. Mu. Mol/L upstream primer and 0.1. Mu. Mol/L downstream primer of target gene, 0.1. Mu. Mol/L-0.4. Mu. Mol/L probe of target gene, 0.1. Mu. Mol/L upstream primer and 0.3. Mu. Mol/L downstream primer of internal standard gene for amplification, 0.05. Mu. Mol/L-0.2. Mu. Mol/L probe of internal standard.

2.2 The PCR enzyme cocktail comprises: 1U/. Mu.l to 5U/. Mu.l Taq DNA polymerase, 1U/. Mu.l to 5U/. Mu.l reverse transcriptase, 1U/. Mu.l to 10U/. Mu.l RNase inhibitor.

2.3 the combination allows the detection of novel coronavirus variants B.1.1.7, B.1.351, B.1.617, B.1.617.1, B.1.617.2, B.1.617.3 or P.1.

3. Primer/probe combination three

3.1 The PCR reaction solution comprises: 2 XPCR reaction buffer, 0.1 mmol/L-0.5 mmol/L deoxyribonucleoside triphosphate, 0.2. Mu. Mol/L-0.6. Mu. Mol/L upstream primer and 0.1. Mu. Mol/L downstream primer of target gene, 0.1. Mu. Mol/L-0.4. Mu. Mol/L probe of target gene, 0.1. Mu. Mol/L upstream primer and 0.3. Mu. Mol/L downstream primer of internal standard gene for amplification, 0.05. Mu. Mol/L-0.2. Mu. Mol/L probe of internal standard.

3.2 The PCR enzyme cocktail comprises: 1U/. Mu.l to 5U/. Mu.l Taq DNA polymerase, 1U/. Mu.l to 5U/. Mu.l reverse transcriptase, 1U/. Mu.l to 10U/. Mu.l RNase inhibitor.

3.3 the combination allows the detection of novel coronavirus variants B.1.1.7, B.1.351, B.1.617 or P.1.

4. Primer/probe combination four

4.1 The PCR reaction solution comprises: 2 XPCR reaction buffer, 0.1 mmol/L-0.5 mmol/L deoxyribonucleoside triphosphate, 0.2. Mu. Mol/L-0.6. Mu. Mol/L upstream primer and 0.1. Mu. Mol/L downstream primer of target gene, 0.1. Mu. Mol/L-0.4. Mu. Mol/L probe of target gene, 0.1. Mu. Mol/L upstream primer and 0.3. Mu. Mol/L downstream primer of internal standard gene for amplification, 0.05. Mu. Mol/L-0.2. Mu. Mol/L probe of internal standard.

4.2 The PCR enzyme cocktail comprises: 1U/. Mu.l to 5U/. Mu.l Taq DNA polymerase, 1U/. Mu.l to 5U/. Mu.l reverse transcriptase, 1U/. Mu.l to 10U/. Mu.l RNase inhibitor.

4.3 the novel coronavirus B.1.617 variant, B.1.617.1 variant, B.1.617.2 variant or B.1.617.3 variant can be detected.

Example 2

The embodiment provides a method for using a detection kit for detecting novel coronaviruses and variants thereof

1. Sample collection:

1.1 samples were used: nasopharyngeal swab and oropharyngeal swab.

1.2 sample collection method: sample collection is performed using a swab in the nasopharynx or oropharynx of the patient, the swab is inserted to the appropriate depth, rotated in situ for a few seconds to absorb the subject's secretions, and the swab is slowly removed with rotation. Immediately after sample collection, the swab is placed into a virus sampling tube containing 1-3 ml of virus preservation solution.

1.3 samples collected should be sent for inspection in time and stored at 4 ℃ for detection within 24 hours.

1.4 sample transportation should be carried out by using a curling to add ice or a foam box to add ice and seal.

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.) may be used and the extraction process should be performed according to the instructions of the commercial kit. The purified nucleic acid can be used for subsequent PCR amplification. Qiagen Viral RNA Mini Kit the specific operation steps are as follows:

2.1.1 into a 1.5ml EP tube was added 560. Mu.l buffer AVL.

2.1.2. Mu.l of sample (or quality control) was added to the EP tube, and the mixture was 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 inside the cap. 560 μl of absolute ethanol was added, vortex mixed, and centrifuged instantaneously to remove water droplets from the lid.

2.1.4 carefully aspirate 630. Mu.l of solution into a QIAamp mini-column (2 ml collection tube), cover 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 the QIAamp mini-column was carefully opened and 500. Mu.l buffer AW1 was added. 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 the QIAamp mini column was carefully opened and 500. Mu.l buffer AW2 was added. The lid was closed and centrifuged at 14000rpm for 3 minutes.

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

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

2.1.9 the QIAamp mini-column was placed in a clean 1.5ml EP tube. The old collection tube containing the filtrate was discarded. The lid was carefully opened and 60 μ buffer AVE was added. The lid was closed, and then allowed to stand at room temperature for 1 minute.

2.1.10 centrifuged at 8000rpm for 1 min. The QIAamp mini-column was discarded, and the EP tube containing the eluate was retained and subsequently used for PCR amplification.

2.2 sample handling directly PCR (Direct PCR)

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

2.2.1 vortex shaking resuspended samples.

2.2.2 into the EP tube, 20. Mu.L of viral nucleic acid releasing agent (Chengdu Fujia) and 2. Mu.L of RNA protecting agent were added and mixed well.

2.2.3 34. Mu.L of sample (or quality control) was added to the aforementioned EP tube containing the nucleic acid releasing agent, and the mixture was blown and mixed uniformly, and allowed to stand at room temperature for 10 minutes, followed by use for PCR amplification.

3. And (3) sample adding:

3.1 novel coronaviruses and variant detection reagents for a variety of different primer/probe combinations were added to PCR tubes. Wherein, the liquid crystal display device comprises a liquid crystal display device,

(1) Taking 15 mu L of a novel coronavirus combined and mutant strain detection reagent and 1.5 mu L of PCR enzyme mixed solution, and respectively adding the novel coronavirus combined and the mutant strain detection reagent and the PCR enzyme mixed solution into a PCR tube 1# and a PCR tube 2 #;

(2) Taking 15 mu L of a novel coronavirus combined with II and a variant detection reagent thereof and 1.5 mu L of a PCR enzyme mixed solution, and respectively adding the two into a PCR tube 1# and a PCR tube 2 #;

(3) Taking 15 mu L of novel coronavirus combined with three and variant detection reagent and 1.5 mu L of PCR enzyme mixed solution, and respectively adding the novel coronavirus combined with three and the variant detection reagent into PCR tube 1#, PCR tube 2# and PCR tube 3#;

(4) 15 mu L of novel coronavirus combined with four and variant detection reagent and 1.5 mu L of PCR enzyme mixed solution are respectively added into PCR tube No. 1;

3.2 the detection reagent of the four primer/probe combinations can be selected to carry out subsequent detection. 15. Mu.L of nucleic acid (2.1 or 2.2) of the treated sample (or quality control product) was added to the PCR amplification system. And (5) covering the PCR tube cover, and waiting for machine loading.

3.3 sample application method example

3.3.1 sample adding method of combination one and combination two

NC-negative quality control; PC-positive quality control

3.3.2 sample adding method of combination III

NC-negative quality control; PC-positive quality control

3.3.3 sample adding method of combination four

NC-negative quality control; PC-positive quality control

Pcr amplification procedure setup and run:

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 opening the instrument and placing the instrument into a PCR tube to be detected.

4.3 amplification procedure was set up. The corresponding fluorescence channels FAM channel (Reporter: FAM, quantum: none), HEX channel (Reporter: HEX, quantum: none), ROX channel (Reporter: ROX, quantum: none), CY5 channel (Reporter: CY5, quantum: none) were selected. Sample editing is performed.

4.4 run amplification program.

4.5 specific amplification procedure was as follows:

5. analysis of results:

5.1 after the reaction is finished, the instrument automatically saves the result, can utilize the software of the instrument to automatically analyze (the starting value, the ending value and the threshold line value of the base line can also be manually adjusted to analyze), and then records 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 reporting as Positive (POS) for samples with Ct value less than or equal to 40; for samples with no Ct value or Ct value >40, a Negative (NEG) is reported. If the internal standard Ct value is >38 or is not displayed, the detection result of the sample is invalid, the reason should be searched and eliminated, and the sample is subjected to repeated tests.

6. Interpretation of the results:

6.1 novel coronavirus and mutant thereof combined by primer/probe and method for judging and reading results of detection reagents

1) ORF1/N (FAM signal of A tube), S69/70 Del (FAM signal of B tube), ACTIN (CY 5 signal) was judged as NEG or POS directly according to 5.2.

2) If the CT value of S253P (HEX signal of A tube) -the CT value of ORF1 (FAM signal of A tube) >6, or if S253P has no CT value, then S253P is NEG. In contrast, S253P is POS.

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

4) If the CT value of P71L (HEX signal of B tube) -the CT value of ORF1 (FAM signal of A tube) >6, or if P71L has no CT value, then P71L is NEG. In contrast, P71L is POS.

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

6.2 novel coronavirus and mutant strain detection reagent result interpretation of primer/probe combination II

1) ORF1/N (FAM signal of A tube), S69/70 Del (FAM signal of B tube), ACTIN (CY 5 signal of A tube) was judged as NEG or POS directly according to 5.2.

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

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

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

6) If the CT value of S253P (CY 5 signal of B tube) -CT value of ORF1 (FAM signal of A tube) >6, or if S253P has no CT value, then S253P is NEG. In contrast, S253P is POS.

6.3 novel coronavirus and mutant strain detection reagent result interpretation of primer/probe combination III

1) ORF1/N (FAM signal of A tube), NSP6 106-108Del (HEX signal of A tube), S69/70 Del (FAM signal of C tube), ACTIN (CY 5 signal) were judged as NEG or POS directly according to 5.2.

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

3) If the CT value of S253P (FAM signal of B tube) -the CT value of ORF1 (FAM signal of a tube) >6, or if S253P has no CT value, then S253P is NEG. In contrast, if S253P is POS.

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

6) If the CT value of P71L (HEX signal of C tube) -the CT value of ORF1 (FAM signal of A tube) >6, or if P71L has no CT value, then P71L is NEG. In contrast, P71L is POS.

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

6.4 novel coronavirus and variant detection reagent result interpretation of primer/probe combination IV

1) ORF1/N (FAM signal), ACTIN (CY 5 signal) was judged as NEG or POS directly according to 5.2.

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

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

Example 3

This example provides a test of novel coronavirus and variants thereof detection reagents prepared from primer/probe combinations.

1. Sample: the RNA1 of the in vitro transcription product of the DNA template (SEQ ID NO. 33) contains RNA fragments of the 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 RNA2 of the in vitro transcription product of the DNA template (SEQ ID NO. 34) contains RNA fragments of ORF3A gene, S gene and E gene.

2. The method comprises the following steps: the procedure of example 2 was followed to dilute the RNA fragments to a concentration of 3pg/ul, to perform parallel detection of the different RNA fragments on an ABI7500 instrument and to perform result analysis and statistics.

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

4. In summary, the reagent (combination one) can detect the ORF1 gene and the N gene of the novel coronavirus and detect the E484K mutation site, the 69/70Del mutation site, the N501Y mutation site, the P71L mutation site and the S253P mutation site of the ORF3A gene of the novel coronavirus at the same time, so as to distinguish corresponding mutants.

Example 4

This example provides a test for the detection of novel coronaviruses and variants thereof prepared from primer/probe combinations II.

1. Sample: the RNA3 of the 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 the RNA4 of the 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 detection results are shown in FIG. 2.

4. In summary, the reagent (combination II) can detect the ORF1 gene and the N gene of the novel coronavirus, and detect the E484Q mutation site, the 69-70Del mutation site, the L452R mutation site, the P71L mutation site, the S253P mutation site and the V82A mutation site of the ORF7A gene of the novel coronavirus, thereby distinguishing the corresponding mutant strains.

Example 5

This example provides an evaluation of clinical performance of a novel coronavirus and variant thereof kit prepared from primer/probe combinations.

1. Sample source: 6 new coronavirus samples that have been validated by third party medical test laboratories.

2. The detection method comprises the following steps: the method of the invention was carried out according to the procedure of example 2, with parallel detection of the same sample on an ABI7500 instrument, and with analysis and statistics of the results.

3. The detection result is specifically as follows:

through the verification of a third-party medical test 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 is consistent with the interpretation result of the reagent.

4. Mutation result analysis: the mutation site detected by the reagent has 100 percent of mutation site accuracy through PCR+Sanger sequencing analysis. See in particular the results of the S HV69-70Del mutation site sequencing and S N501Y mutation site sequencing in FIG. 3.

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

Example 6

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

1. Sample source: 10 new coronavirus samples have been validated by third party medical test laboratories.

3. The detection result is specifically as follows:

the third-party medical test laboratory verifies that the sample variant in the embodiment is consistent with the interpretation result of the reagent.

4. Mutation result analysis: the mutation site detected by the reagent has 100 percent of mutation site accuracy through PCR+Sanger sequencing analysis. See for details NSP6 106/107/108Del mutation site sequencing results, S E484K mutation site sequencing results, ORF3A S253P mutation site sequencing results, ORF7A V A mutation site sequencing results, S L452R mutation site sequencing results, S HV69-70Del mutation site sequencing results, E P L mutation site sequencing results, S E484Q mutation site sequencing results in FIG. 4.

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

The foregoing description is only of the preferred embodiments of the invention and is not intended to limit the invention. Various modifications and variations of the present invention will be apparent to those skilled in the art. The partial modification of the primer probe sequence, the combination of the primer probe, the fluorescent marking of the probe, the use of the probe complementary sequence and the like are all considered to be within the spirit and principle of the invention. Any modifications, equivalent substitutions, improvements, etc. which are within the spirit and principles of the present invention are intended to be included within the scope of the present invention.

Sequence listing

<110> Chengdu peak biotechnology Co., ltd

<120> a primer, a probe composition and a kit for detecting novel coronavirus and variants thereof

<160> 36

<170> SIPOSequenceListing 1.0

<210> 1

<211> 21

<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

<210> 5

<211> 29

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 5

gtgattagac ttcctaaaca atctattcc 29

<210> 6

<211> 37

<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 coronaviruses and variants thereof, comprising primers and probes specific for the ORF1ab gene, the N gene and the internal reference gene of SARS-CoV-2, characterized by further comprising:

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

the primer and the probe composition are used for detecting mutation sites when detecting the novel coronavirus, and can distinguish the novel coronavirus B.1.351 variant, the novel coronavirus B.1.1.7 variant, the novel coronavirus B.1.617 variant and the novel coronavirus P.1 variant;

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 S gene N501Y mutation site 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 NSP6 gene 106/107/108del mutation site 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 mutation site of the E gene P71L 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 the 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 mutation site of the ORF3A gene S253P 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 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;

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;

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 of the ACTIN gene is shown as SEQ ID NO.31, and a probe of the ACTIN gene is shown as SEQ ID NO. 32;

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

the 5' end of the probe of the S gene L452R, E484K, E484Q, N501Y mutation site adopts the same second fluorescent group;

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

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

2. The primer and probe composition for detecting novel coronaviruses and variants thereof as claimed in claim 1, wherein:

the 5 'ends of 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 'end of the probe of the S gene L452R, E484K, E484Q, N501Y mutation site adopts ROX fluorescent groups, and the 3' end adopts BHQ2 quenching groups;

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

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

3. The primer and probe composition for detecting novel coronaviruses and variants thereof according to claim 1 or 2, wherein the primer and probe combinations of each of the genes and mutation sites form different mixed solutions;

in one mixed solution, probes of the ORF1ab gene and the N gene exist at the same time and are different from fluorescent groups at the 5' end of probes of other genes or mutation sites in the mixed solution;

the fluorescent groups at the 5' end of each probe in each other mixed solution are different;

each mixture was used to perform multiple fluorescent PCR reactions using one PCR tube.

4. The primer and probe composition for detecting novel coronaviruses and variants thereof as claimed in claim 3, wherein the number of said mixed solution is 1-3.

5. The primer and probe composition for detecting novel coronaviruses and variants thereof according to claim 3, wherein the mixed solution is a PCR reaction solution A and a 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 liquid B contains an S gene 69-70Del mutation site, an E gene P71L mutation site, an N501Y mutation site and a probe;

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

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

6. The primer and probe composition for detecting novel coronaviruses and variants thereof according to claim 3, wherein the mixed solution is a PCR reaction solution C and a 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 internal 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 coronaviruses, and novel coronavirus B.1.1.7 variants, B.1.351 variants, B.1.617 variants, B.1.617.1 variants, B.1.617.2 variants, B.1.617.3 variants or P.1 variants.

7. The primer and probe composition for detecting novel coronaviruses and variants thereof according to claim 3, wherein the mixed solution is a PCR reaction solution E, 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 an ORF3A gene S253P mutation site, an ORF7A gene V82A mutation site and an 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;

primer and probe of internal reference gene are contained in the PCR reaction liquid E, F and/or G;

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

8. The primer and probe composition for detecting novel coronaviruses and variants thereof according to claim 3, wherein the mixed solution 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.

9. A kit for detecting novel coronaviruses and variants thereof, comprising the primer and probe composition for detecting novel coronaviruses and variants thereof according to any one of claims 1 to 8.