A primer set and probe for detecting a novel coronavirus 7a subgene, and application and product thereof

Abstract

The application discloses a primer set and a probe for detecting a novel coronavirus 7a subgene, and application and products thereof, and relates to the technical field of biology. The primer set comprises an upstream primer shown as SEQ ID NO. 4 and a downstream primer shown as SEQ ID NO. 2; and the nucleotide sequence of the probe is shown as SEQ ID NO. 3. According to transcriptome sequencing analysis, it is found that the expression amounts of N, E and 7a subgenes are relatively high in the virus replication process. The application provides the primer set and the probe for the novel coronavirus 7a subgene, and realizes rapid detection of the novel coronavirus subgene by using a fluorescent RT-RAA technology, so as to provide a reference for judging the infectivity of a SARS-CoV-2 clinical sample.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a primer set and probe for detecting a novel coronavirus 7a subgene, and application and product thereof. BACKGROUND

[0002] COVID-19 is an acute infectious disease caused by SARS-CoV-2. The pathogenic mechanism of novel coronavirus infection in humans is not fully elucidated. After the human body is infected with the virus, viral nucleic acid can be detected for up to 2 months or so, and anal swab detection is positive for a longer time. At present, the discharge and isolation criteria for COVID-19 patients are based on nucleic acid test negative. However, it is not certain whether the viral nucleic acid that can be detected by PCR technology after several weeks of clinical recovery is infectious. There are also many cases of “re-positive” patients in clinical practice, but there are no cases of transmission due to re-positive, so whether these re-positive cases are infectious is unknown. Previously, it has been reported that SARS-CoV-2 strains were successfully isolated from frozen food outer packaging samples that were nucleic acid positive, which indicates that environmental samples with nucleic acid positive also have infectivity. However, the method of virus isolation and culture to determine the infectivity of “re-positive” cases or environmental samples requires experiments to be carried out in a biosafety level 3 (BSL-3) laboratory, and the operation is complex, time-consuming, and has poor sensitivity, which affects the isolation and discharge time of patients, and also increases the economic burden of the government and hospitals. Therefore, a method for rapidly detecting whether SARS-CoV-2 nucleic acid positive samples are infectious has important practical significance.

[0003] SARS-CoV-2 is similar to other coronaviruses, which is an enveloped, single-stranded positive-sense RNA virus with a genome of about 30 kb in length. The 5'UTR region of the SARS-CoV-2 genome contains a ~70 nt leader sequence (5'-leader), and there are many transcription regulatory sequences (TRSs) adjacent to each open reading frame (ORF), including the body TRS (TRS-B) and the 5'-leader TRS (TRS-L). After the virus enters the host cell, it first forms a complementary negative-strand RNA using the positive-strand RNA as a template, and then uses the generated negative-strand RNA as a template for transcription to produce positive-strand genomic RNA (gRNA). However, when the genome replicates to produce a negative strand, the RNA-dependent RNA polymerase (RdRP) pauses and jumps to the TRS-L when passing through the TRS-B. This discontinuous replication results in the fusion of the 5'-leader and the body sequence to splice into negative-strand RNAs of different lengths, and the transcription of the negative-strand RNAs produces various sgRNAs. These sgRNAs encode viral structural proteins (S, E, M, N), as well as auxiliary proteins (3a, 6, 7a, 7b, 8, and 10). Although the functions of sgRNAs in the replication and pathogenesis of SARS-CoV-2 need to be further elucidated, sgRNAs are only transcribed in infected cells and are not packaged into viral particles to be released from the cells, so the detection of sgRNAs can indicate the presence of active infection. There are reports that sgRNAs can be detected in the early stages of infection and not detected in the later stages of infection, which indicates that the presence of sgRNAs is a marker of active replication of SARS-CoV-2, rather than residual viral RNA, so sgRNAs detection can be used to determine whether a SARS-CoV-2 nucleic acid positive sample is infectious. SUMMARY

[0004] The purpose of the present application is to provide a primer set and probe for detecting the 7a subgene of the novel coronavirus and its application and product, in order to solve the problems existing in the prior art. According to transcriptome sequencing analysis, it is found that the expression amounts of N, E and 7a subgenes are higher in the virus replication process. The present application aims to provide a primer and probe combination for the 7a subgene of the novel coronavirus, and to realize rapid detection of the subgene of the novel coronavirus by using fluorescent RT-RAA technology, thereby providing a reference for judging the infectivity of SARS-CoV-2 clinical samples.

[0005] To achieve the above-mentioned purpose, the present application provides the following solutions:

[0006] The application provides a primer set and a probe for detecting a novel coronavirus 7a subgene, wherein the primer set comprises an upstream primer shown as SEQ ID NO. 4 and a downstream primer shown as SEQ ID NO. 2; and the nucleotide sequence of the probe is shown as SEQ ID NO. 3.

[0007] Further, the 5' end of the probe is labeled with a FAM fluorescent group, and the 3' end is labeled with a BHQ1 fluorescent group.

[0008] The application also provides application of the primer set and the probe in preparation of a kit or reagent for detecting the novel coronavirus 7a subgene.

[0009] The application also provides a product for fluorescent RT-RAA detection of the novel coronavirus 7a subgene, which comprises the primer set and the probe.

[0010] Further, the product comprises a kit or a reagent.

[0011] The application discloses the following technical effects:

[0012] (1) It is of great significance to determine whether the virus carried by long-term positive or "re-positive" novel coronavirus infected people or in the environment is infectious for the treatment and management of cases and prevention and control. The traditional virus isolation and culture has low sensitivity and long time consumption. The subgene can be detected by the fluorescent RT-RAA method to quickly respond to the virus infectivity.

[0013] (2) The expression amount of each gene in the novel coronavirus subgenome is quite different, and the application detects the 7a subgene with high expression amount, thereby improving the sensitivity of detection.

[0014] (3) The application analyzes the novel coronavirus subgenome through bioinformation analysis, selects the conservative region of the novel coronavirus 7a subgene to design the primer probe, and ensures that the subgene of the current novel coronavirus genotype or evolutionary branch can be detected. In addition, the primer probe is verified for specificity with other nine kinds of respiratory virus genomes and subgenomes of human infection, and has good specificity. BRIEF DESCRIPTION OF DRAWINGS

[0015] In order to more clearly illustrate the technical solutions in the embodiments of the application or the prior art, the following will briefly introduce the drawings needed in the embodiments. Obviously, the drawings in the following description only constitute some embodiments of the application, and for those skilled in the art, other drawings can also be obtained without creative labor on the basis of these drawings.

[0016] Figure 1 The amplification curves of the novel coronavirus positive samples before and after primer optimization are compared;

[0017] Figure 2 For the sensitivity of the subgenomic fluorescent RT-RAA detection method of the novel coronavirus, the 1:1000 dilution SARS-CoV-2 cell culture nucleic acid was quantified by digital PCR method (ddPCR) (20 copies / μL), and the fluorescent RT-RAA detection of 1:1000 dilution SARS-CoV-2 cell culture nucleic acid could effectively amplify;

[0018] Figure 3 For the specificity of the subgenomic fluorescent RT-RAA detection method of the novel coronavirus, the frame line represents the nucleic acid of HCoV-NL63, HCoV-OC43, HCoV-HKU, HCoV-229, influenza A virus, influenza B virus, respiratory syncytial virus, rhinovirus, and parainfluenza virus, respectively, and none of them is specifically amplified. DETAILED DESCRIPTION

[0019] The various illustrative embodiments of the present application will now be described in detail below. This detailed description is merely intended to teach a person skilled in the art further how to make and use the present application and is not intended to limit the scope of the present application. Therefore, the present application should be understood to include any variations of the embodiments described herein that come within the scope of the present application. Those skilled in the art will readily recognize from this disclosure that alternative embodiments of the present application can be devised without departing from the scope of the present application. Accordingly, the present application should not be limited to the embodiments described herein but should be given the full scope defined by the language of the claims.

[0020] It should be understood that the terms used in the present application merely describe particular embodiments and are not intended to limit the present application. In addition, for numerical ranges in the present application, it should be understood that each intermediate value between the upper limit and the lower limit of the range is also specifically disclosed. Each smaller range within any stated range or within any stated intermediate value, and any other stated value or intermediate value within the stated range is also included in the present application. The upper limit and the lower limit of these smaller ranges can be included or excluded independently from the range.

[0021] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present application pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, the preferred methods and materials are described herein. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials in connection with which the documents are cited. In the case of conflict between any document incorporated by reference and the present specification, the present specification will control.

[0022] Various modifications and changes can be made to the specific embodiments of the present application described herein without departing from the scope or spirit of the application. Other embodiments of the application will be apparent to those of ordinary skill in the art from the description and examples herein. The description and examples are illustrative only.

[0023] As used herein, the terms “comprises,” “comprising,” “includes,” “including,” “has,” “having,” and the like are open-ended terms that are intended to be synonymous, and are generally used in the sense of “including, but not limited to.”

[0024] Primer and probe design of Example 1

[0025] The novel coronavirus full genome sequences were downloaded from the GISAID database, and sequence alignment was performed using the MAFFT software. The 5' UTR region leader sequence and the 5' end of the 7a gene were extracted to constitute the primer and probe design template. The fluorescent RT-RAA primers and Taqman probes were designed using Primer express 3.0 and Primer 5.0 software. The upstream primer was designed to fall into the leader region, and the downstream primer was designed to fall into the 7a gene region. The probe sequence can fall into either region or span two regions. This design can amplify the 7a subgene, avoiding the synchronous amplification of 7a gene RNA. The designed primers and probes were compared using NCBI BLAST, which excluded homology with SARS, MERS, 229E, OC43, NL63, HKU1 coronaviruses and other microorganisms, and human genome. The 5' end of the Taqman probe was labeled with FAM fluorescent group, and the 3' end was labeled with BHQ1 fluorescent group. The primer and probe sequences are shown in Table 1 SEQ ID NO. 1-3:

[0026] Table 1 Primer and probe sequences

[0027]

[0028] Optimization of primers of Example 2

[0029] In order to achieve the best amplification effect, single base point mutation was performed on the upstream primer sequence, and the base was mutated from C to A (SEQ ID NO. 4), so that the difference between the GC content and Tm value of the upstream and downstream primers was smaller, the mismatch rate was reduced, and the amplification efficiency was improved. The nucleic acid extracted from the positive clinical samples of the new crown (2020-COVID-19-1 and 2020-COVID-19-2) was used to compare the effects of the primers before and after optimization (Example 2), and the results showed that after point mutation, the curve of the primer amplification had an earlier peak time, and the final fluorescence value was larger, achieving better amplification effect. Figure 1

[0030] Establishment and optimization of fluorescent RT-RAA detection method of Example 3

[0031] Qiagen company ​The Viral RNA Mini Kit extracted RNA from 200 μL of inactivated novel coronavirus cell culture medium as a template for amplification. With the template concentration fixed, the optimal primer, probe concentration, and reaction conditions were determined based on the amplification curve shape, peak time, and fluorescence signal intensity using a QT-F1620 nucleic acid amplification fluorescence detector from Qitian Gene. The optimized fluorescent RT-RAA reaction system was as follows: 25 μL buffer IV, 2.1 μL (10 μmol / L) upstream primer (SEQ ID NO.4), 2.1 μL (10 μmol / L) downstream primer, 0.6 μL (10 μmol / L) probe, and 12.7 μL RNase-free sterile water. The optimized amplification reaction conditions were: pre-amplification at 39℃ for 7 min, followed by amplification at 39℃ for 30 min, with real-time observation of the results.

[0032] Example 4 Sensitivity Evaluation

[0033] Nucleic acid extracted from SARS-CoV-2 cell cultures was used as templates for ddPCR detection at dilutions of 1:50, 1:100, 1:500, and 1:1000. The results showed that... Figure 2 The copy number of SARS-CoV-2 cell culture nucleic acid at a 1:1000 dilution was 20 copies / μL. The SARS-CoV-2 cell culture nucleic acid at the above dilution was then subjected to fluorescent RT-RAA detection. The results showed that the 1:1000 dilution of nucleic acid began to amplify at approximately 2 minutes, and the final fluorescence value was 11085 after 30 minutes of amplification at 39℃. This indicates that the fluorescent RT-RAA detection method can effectively amplify samples with a nucleic acid concentration of 20 copies / μL.

[0034] Example 5 Specificity Evaluation

[0035] Nucleic acid was extracted from SARS-CoV-2 cell cultures and sputum samples from patients infected with other respiratory pathogens such as HCoV-NL63, HCoV-OC43, HCoV-HKU, HCoV-229, influenza A virus, influenza B virus, respiratory syncytial virus, rhinovirus, and parainfluenza virus as templates. Fluorescent RT-RAA detection was performed according to the optimized method in Example 2. The results showed ( Figure 3 Only SARS-CoV-2 cell culture nucleic acid showed a specific amplification curve, while nucleic acids extracted from HCoV-NL63, HCoV-OC43, HCoV-HKU, HCoV-229, influenza A virus, influenza B virus, respiratory syncytial virus, rhinovirus, and parainfluenza virus infection cases did not show amplification curves, indicating that the established RT-RAA detection method has good specificity.

[0036] Example 6 Consistency Evaluation

[0037] The clinical samples of 104 cases of clinically diagnosed SARS-CoV-2 infection in 2020 were detected by Real-time PCR method (reference Roman Victor M Corman, Wolfgang Guggemos, et al. Virological assessment of hospitalized patients with COVID-2019 [J]. Nature, 2020, 581 (7809): 465-469) and the subgenomic sgRNAs were detected by the optimized fluorescent RT-RAA method of Example 2 for consistency evaluation. The results showed (Table 2) that 17 cases were positive and 79 cases were negative by both Real-time PCR and fluorescent RT-RAA detection; 2 cases were positive by Real-time PCR but negative by fluorescent RT-RAA detection, and 6 cases were positive by fluorescent RT-RAA but negative by Real-time PCR. The consistency of the two detection methods was good (κ = 0.762, P < 0.001).

[0038] Table 2 Comparison of Real-time PCR and RT-RAA detection of sgRNAs in SARS-CoV-2 clinical samples

[0039]

[0040]

[0041] The above-described embodiments are only to describe the preferred modes of the present application, and not to limit the scope of the present application. Without departing from the design spirit of the present application, various modifications and improvements to the technical solutions of the present application made by those of ordinary skill in the art shall fall within the protection scope determined by the claims of the present application. SEQUENCE LISTING <110> Jiangsu Center for Disease Control and Prevention (Jiangsu Provincial Institute for Public Health) <120> Primer set and probe for detecting 7a subgene of novel coronavirus, application and product thereof <160> 4 <170> SIPOSequenceListing 1.0 <210> 1 <211> 31 <212> DNA <213> Artificial Sequence <400> 1 atggtgaatt gccctcgtat gttccagaag a 31 <210> 2 <211> 32 <212> DNA <213> Artificial Sequence <400> 2 caaccaactt tcgatctctt gtagatctgt tc 32 <210> 3 <211> 47 <212> DNA <213> Artificial Sequence <400> 3 tagtgataaa gctcacaagt agcgagtgtt atcagtgcca agaaaag 47 <210> 4 <211> 31 <212> DNA <213> Artificial Sequence <400> 4 atggtgaatt gccatcgtat gttccagaag a 31

Claims

1. A primer set and probe for fluorescent RT-RAA detection of novel coronavirus, characterized in that, The primer set comprises an upstream primer as shown in SEQ ID NO. 4 and a downstream primer as shown in SEQ ID NO. 2; the nucleotide sequence of the probe is shown in SEQ ID NO.

3.

2. The primer set and probe according to claim 1, characterized in that, The 5' end of the probe is labeled with a FAM fluorescent group, and the 3' end is labeled with a BHQ1 fluorescent quenching group.

3. Use of the primer set and the probe according to claim 1 or 2 in the preparation of a kit or reagent for detecting the novel coronavirus.

4. A product for detecting a novel coronavirus by fluorescent RT-RAA, characterized in that, The product comprises the primer set and the probe according to claim 1 or 2.

5. The product of claim 4, wherein, The product comprises a kit or a reagent.

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