CN113549709A

CN113549709A - Primer pair, probe and kit for detecting SARS-CoV-2 by utilizing nested RPA technology and application thereof

Info

Publication number: CN113549709A
Application number: CN202010337404.5A
Authority: CN
Inventors: 黄婉秋; 黄健
Original assignee: Individual
Current assignee: Individual
Priority date: 2020-04-26
Filing date: 2020-04-26
Publication date: 2021-10-26

Abstract

The invention discloses a primer pair, a probe and a kit for detecting SARS-CoV-2 novel coronavirus, wherein the kit comprises the primer pair and the probe which are designed aiming at any one or more than two genes in the following SARS-CoV-2 novel coronavirus complete gene sequence: ORF1ab, S gene, ORF6, ORF7a, ORF7b, ORF8, N gene, ORF 10. The invention discloses a reagent box for detecting SARS-CoV-2 new type coronavirus, which uses a nest type RPA method to detect SARS-CoV-2 virus nucleic acid, can detect SARS-CoV-2 nucleic acid segment of 1copy/ul at least, has ultrahigh sensitivity, provides new technical support for early discovery, early isolation and early treatment of new type coronavirus SARS-CoV-2, and has good application prospect.

Description

Primer pair, probe and kit for detecting SARS-CoV-2 by utilizing nested RPA technology and application thereof

Technical Field

The invention belongs to the technical field of biological engineering, and particularly relates to a primer pair, a probe and a kit for detecting SARS-CoV-2 based on a nested RPA technology, and application thereof.

Background

The new type coronavirus infectious pneumonia (COVID-19) is characterized by long latent period, strong infectivity and high death rate. The epidemic spreads rapidly, causing a huge social hazard. The new coronarism COVID-19 is caused by a new type of coronavirus SARS-CoV-2 infection.

Coronavirus (CoV), an enveloped single-stranded RNA virus, is widely transmitted in humans, and other mammals and birds, and can cause respiratory, intestinal, hepatic and nervous system diseases. 7 coronaviruses are known to cause disease in humans, 4 of which, such as Cov-229E, Cov-OC43, Cov-NL63 and Cov-HKU1, are prevalent in the human population and often cause common cold symptoms. In addition, the 3 coronaviruses SARS-CoV, MERS-CoV and SARS-CoV-2 are highly dangerous and can cause severe pneumonia and even death.

Early diagnosis and early treatment are the key in the prevention and control measures of the new coronavirus. The virus nucleic acid detection is an important means for quickly diagnosing COVID-19, and the current nucleic acid detection method mainly adopts a fluorescent quantitative PCR (RT-qPCR) technology.

However, the RT-qPCR-based virus nucleic acid detection technology has some difficult problems in clinical application, and false negative results are reported in hospitals in various cities in China. Moreover, some patients were found to be negative with two nucleic acid tests after regular treatment, whereas their nucleic acid test results were again positive 14 days after isolation and observation. This false negative result greatly increases the complexity of the control of COVID-19 epidemic.

Viral nucleic acid testing, from the time a sample is taken from a patient to the completion of a test report, proceeds through a number of steps including sample collection, sample storage, sample transport to a testing laboratory, virus inactivation, cell lysis, nucleic acid extraction, testing and reporting of the test report. Any one of these steps may be somewhat problematic and may lead to false negative results. Wherein, the detection technology with high sensitivity, high specificity and high repeatability is very important for improving the detection quality of SARS-CoV-2 nucleic acid and reducing the false negative rate of experiments.

Recombinase Polymerase Amplification (RPA) is based on a Recombinase Polymerase-mediated Amplification principle, simulates an enzyme reaction process of in vivo DNA replication, depends on specific enzymes and protein combinations (Recombinase, single-stranded binding protein and DNA Polymerase) to perform specific Amplification on a DNA template, can realize rapid specific Amplification (25-42 ℃ for 5-30min) under the condition of approaching body temperature, is used as an isothermal technology to reduce the dependence on high-precision expensive instruments, stable power supply facilities and high-level laboratories, can be completed by only one thermostatic device, can judge whether Amplification products exist or not by a fluorescence analysis device, has the characteristic of simpler facility and operation requirements, and is applied to multiple fields of life science research, medical detection, agriculture, food safety, transgenic detection and the like.

In order to prevent and control the spread of COVID-19 epidemic situation, a novel detection technology with high sensitivity and high speed is urgently needed to be developed.

Disclosure of Invention

The invention aims to solve the technical problem of the lack of a rapid and accurate SARS-CoV-2 detection kit at present, and provides a primer pair, a probe and a kit for detecting SARS-CoV-2 based on a nested RPA technology, wherein the kit has ultrahigh sensitivity and can rapidly, accurately and specifically detect SARS-CoV-2.

In order to solve the technical problems, the invention is realized by the following technical scheme:

in one aspect of the invention, a primer pair for detecting SARS-CoV-2 novel coronavirus by nested RPA is provided, wherein the primer pair is designed aiming at any one or more than two genes in the following SARS-CoV-2 novel coronavirus complete gene sequence: ORF1ab, S gene, ORF6, ORF7a, ORF7b, ORF8, N gene, ORF 10.

Preferably, the primer pair is a primer pair for amplifying SEQ ID NO: 1 to SEQ ID NO: 8 in the sequence listing.

The amplification of SEQ ID NO: 1 comprises the following components: the outer primer pair SEQ ID NO: 10 and SEQ ID NO: 11, inner primer pair SEQ ID NO: 12 and SEQ ID NO: 13.

the amplification of SEQ ID NO: 2 comprises the following components: the outer primer pair SEQ ID NO: 15 and SEQ ID NO: 16, inner primer pair SEQ ID NO: 17 and SEQ ID NO: 18.

the amplification of SEQ ID NO: 3 comprises the following components: the outer primer pair SEQ ID NO: 20 and SEQ ID NO: 21, inner primer pair SEQ ID NO: 22 and SEQ ID NO: 23.

the amplification of SEQ ID NO: 4 comprises the following components: the outer primer pair SEQ ID NO: 25 and SEQ ID NO: 26, inner primer pair SEQ ID NO: 27 and SEQ ID NO: 28.

the amplification of SEQ ID NO: 5 comprises the following components: the outer primer pair SEQ ID NO: 30 and SEQ ID NO: 31, inner primer pair SEQ ID NO: 32 and SEQ ID NO: 33.

the amplification of SEQ ID NO: 6 comprises the following primer pairs: the outer primer pair SEQ ID NO: 35 and SEQ ID NO: 36, inner primer pair SEQ ID NO: 37 and SEQ ID NO: 38.

the amplification of SEQ ID NO: 7 comprises the following primer pairs: the outer primer pair SEQ ID NO: 40 and SEQ ID NO: 41, inner primer pair SEQ ID NO: 42 and SEQ ID NO: 43.

the amplification of SEQ ID NO: 8 comprises the following primer pairs: the outer primer pair SEQ ID NO: 45 and SEQ ID NO: 46, inner primer pair SEQ ID NO: 47 and SEQ ID NO: 48.

in another aspect of the present invention, there is also provided a probe for detecting SARS-CoV-2 novel coronavirus by nested RPA, wherein the probe is specifically designed for any one or more than two genes in the following SARS-CoV-2 novel coronavirus complete gene sequence: ORF1ab, S gene, ORF6, ORF7a, ORF7b, ORF8, N gene, ORF 10.

Preferably, the probe hybridizes to SEQ ID NO: 1 to SEQ ID NO: 8 in the presence of a primer, and specifically hybridizing a part of the sequence of any one of the gene fragments.

More preferably, the probe comprises:

and SEQ ID NO: 1 gene fragment having the sequence of SEQ ID NO: 9;

and SEQ ID NO: 2 gene fragment having the sequence of SEQ ID NO: 14;

and SEQ ID NO: 3, a partial sequence of the gene fragment specifically hybridizes with a sequence of SEQ ID NO: 19;

and SEQ ID NO: 4 gene fragment having the sequence of SEQ ID NO: 24;

and SEQ ID NO: 5 gene fragment having the sequence of SEQ ID NO: 29;

and SEQ ID NO: 6 gene fragment having the sequence of SEQ ID NO: 34;

and SEQ ID NO: 7 gene fragment having the sequence of SEQ ID NO: 39;

and SEQ ID NO: 8 gene fragment having the sequence of SEQ ID NO: 44, or a fragment thereof.

In another aspect of the invention, a kit for detecting SARS-CoV-2 is also provided, which comprises the above primer pair, and the probe corresponding to the primer pair and other necessary matched buffer solution and enzyme.

Preferably, the kit comprises SEQ ID NO: 29, and a probe that amplifies the sequence shown in SEQ ID NO: 5, the primer pair comprises: the outer primer pair SEQ ID NO: 30 and SEQ ID NO: 31, inner primer pair SEQ ID NO: 32 and SEQ ID NO: 33.

preferably, the kit comprises SEQ ID NO: 39, and a probe that amplifies the sequence shown in SEQ ID NO: 7, the primer pair comprises: the outer primer pair SEQ ID NO: 40 and SEQ ID NO: 41, inner primer pair SEQ ID NO: 42 and SEQ ID NO: 43.

in another aspect of the invention, the application of the above-mentioned kit in the preparation of products for detecting SARS-CoV-2 new type coronavirus is also provided.

In another aspect of the present invention, there is also provided a method for detecting viruses with nested RPAs, comprising the steps of:

carrying out a first RPA reaction by using a pair of external primers and virus nucleic acid as a template to amplify a first segment of a target gene;

using a pair of inner primers to amplify a second segment of the target gene by taking the amplified first segment of the target gene as a template and carrying out a second RPA reaction under the condition of adding a signal probe;

and detecting the probe signal to obtain the detection result of the virus nucleic acid.

The signal probe is a probe with a signal label, and is usually a probe with a fluorescein label, such as a FAM fluorescein-labeled probe.

In another aspect of the present invention, there is also provided a primer set for detecting SARS-CoV-2 novel coronavirus, wherein the primer set is designed for any one or more than two of the following SARS-CoV-2 novel coronavirus complete gene sequences: ORF1ab, S gene, ORF6, ORF7a, ORF7b, ORF8, N gene, ORF 10.

Preferably, the primer pair is designed aiming at three genes of SARS-CoV-2 novel coronavirus ORF7a, ORF7b and ORF8, and is used for amplifying the nucleotide sequence shown in SEQ ID NO: 5.

In another aspect of the present invention, there is also provided a probe for detecting SARS-CoV-2 novel coronavirus, wherein the probe is designed for any one or more than two genes of the following SARS-CoV-2 novel coronavirus complete gene sequence: ORF1ab, S gene, ORF6, ORF7a, ORF7b, ORF8, N gene, ORF 10.

Preferably, the probe hybridizes to SEQ ID NO: 5, and specifically hybridizing partial sequences of the gene segments shown in the specification.

The invention discloses a reagent box for detecting SARS-CoV-2 new type coronavirus, which adopts a nest type RPA method to detect SARS-CoV-2 virus nucleic acid, can detect SARS-CoV-2 nucleic acid segment of 1copy/ul at least, has ultrahigh sensitivity, provides technical support for early discovery, early isolation and early treatment of new type coronavirus SARS-CoV-2, and has very good application prospect.

Drawings

The present invention will be described in further detail with reference to the accompanying drawings and specific embodiments.

FIG. 1 is a schematic diagram of the design of a primer in example 1 of the present invention;

FIG. 2 is a histogram of LOD of 16 pairs of primers of example 1 of the present invention;

FIG. 3 is a graph showing the results of the sensitivity of nested RPA detection using the primer pairs and probes of Fragment 5 and Fragment 7 of example 2 of the present invention;

FIG. 4 is a diagram showing the result of nested RPA detection of the repeat worker physical examination sample according to example 3 of the present invention;

FIG. 5 is a high throughput sequencing graph of amplification products that are positive in nested RPA assay according to example 3 of the present invention.

Detailed Description

The spreading of new coronavirus infection pneumonia (COVID-19) caused by SARS-CoV-2 is devastating worldwide and the situation is very severe. The SARS-CoV-2 nucleic acid detection is the main means of COVID-19 diagnosis, and the invented ultra-high sensitivity detection new technology provides new hope for early detection, early isolation and early treatment of SARS-CoV-2.

The invention provides a Nest type RPA (Nest type polymerase amplification, nestRPA) concept, and develops an ultra-high sensitivity SARS-CoV-2 nucleic acid detection technology based on a fluorescence detection analyzer, and SARS-CoV-2 nucleic acid fragments of 1copy/ul can be detected under laboratory conditions. Clinical sample verification finds that: the nestRPA technology can detect samples with positive results of qPCR detection 100%, wherein 1 sample with positive results of qPCR detection still has positive results after 11 times of 10-fold serial dilution, and the samples have negative results after the third dilution. The nestRPA amplification product was verified by high throughput sequencing and 100% detected the target sequence. Meanwhile, 101 qPCR samples with negative detection results are positive, and 32.67 percent (33/101) of the samples detected by the nested RPA of the invention are positive. In addition, 36 samples from multiple hospitalizations of COVID-19 patients that were negative in qPCR were tested, and nestRPA tested positive in 91.67% (33/36) of the samples. 32 throat swab samples of the double-worker examiners who were negative in the result of the qPCR detection by the method of the present invention of the nestRPA showed that 12.5% (4/32) of the samples were positive.

EXAMPLE 1 design and screening of primers and probes for the RPA detection of SARS-CoV-2 novel coronavirus

1. Design of primers and probes

8 sets of primers and probes for RPA detection (see Table 1 and FIG. 1 below) and 8 sets of primers and probes (Probe 1-Probe 8) are designed aiming at conserved regions ORF1ab, S gene, ORF6, ORF7a, ORF7b, ORF8, N gene and ORF10 of SARS-CoV-2 gene sequence (GenBank: NC-045512.2) and correspond to SEQ ID NO: 1-8, and eight amplified fragments of fragments 1-8: fragment 1 (fragment.1, located in ORF1ab, SEQ ID NO: 1), Fragment 2 (fragment.2, located in the S gene, SEQ ID NO: 2), Fragment 3 (fragment.3, spanning ORF6, two genes of ORF7a, SEQ ID NO: 3), Fragment 4 (fragment.4, located in ORF7a, SEQ ID NO: 4), Fragment 5 (fragment.5, spanning ORF7a, ORF7b, three genes of ORF8, SEQ ID NO: 5), Fragment 6 (fragment.6, spanning ORF8, two genes of N, SEQ ID NO: 6), Fragment 7 (fragment.7, located in the N gene, SEQ ID NO: 7), Fragment 8 (fragment.8, located in the N gene, SEQ ID NO: 8).

TABLE 1 primer and Probe sequences for RPA detection

Probe 2	5‘-TGTAATGGTGTTGAAGGTTTTAATTGTTAC/i6FAMdT/C/idSp//iBHQ1dT/CCTTTACAATCATAT-3’
		F2-1	5‘-AATCTATCAGGCCGGTAGCACACCTTGTAATGGTG-3’
F2-2	5‘-ACTGAAATCTATCAGGCCGGTAGCACACCTTGTAA-3’
		F2-3	5‘-TTTCAACTGAAATCTATCAGGCCGGTAGCACACCT-3’
F2-4	5‘-AGATATTTCAACTGAAATCTATCAGGCCGGTAGCA-3’
		F2-5	5‘-GAGAGAGATATTTCAACTGAAATCTATCAGGCCGG-3’
F2-6	5‘-CTTTTGAGAGAGATATTTCAACTGAAATCTATCAG-3’
		F2-7	5’-CAAACCTTTTGAGAGAGATATTTCAACTGAAATCT-3‘
F2-8	5’-AATCTCAAACCTTTTGAGAGAGATATTTCAACTGA-3‘
		F2-9	5’-AGTCTAATCTCAAACCTTTTGAGAGAGATATTTCA-3‘
F2-10	5’-TAGGAAGTCTAATCTCAAACCTTTTGAGAGAGATA-3‘
		R2-1	5'-GGTTGGTAACCAACACCATTAGTGGGTTGGAAACC-3'
R2-2	5'-TGTATGGTTGGTAACCAACACCATTAGTGGGTTGG-3'
		R2-3	5'-TACTCTGTATGGTTGGTAACCAACACCATTAGTGG-3'
R2-4	5'-ACTACTACTCTGTATGGTTGGTAACCAACACCATT-3'
		R2-5	5'-AAAGTACTACTACTCTGTATGGTTGGTAACCAACA-3'
R2-6	5'-AAAAGAAAGTACTACTACTCTGTATGGTTGGTAAC-3'
		R2-7	5'-AGTTCAAAAGAAAGTACTACTACTCTGTATGGTTG-3'
R2-8	5'-GTAGAAGTTCAAAAGAAAGTACTACTACTCTGTAT-3'
		R2-9	5'-TGCATGTAGAAGTTCAAAAGAAAGTACTACTACTC-3'
R2-10	5'-GCTGGTGCATGTAGAAGTTCAAAAGAAAGTACTAC-3'

Probe 3	5'-TAATTATTATGAGGACTTTTAAAGTTTCCA/i6FAMdT//idSp//iBHQ1dT/GGAATCTTGATTACA-3'
		F3-1	5'-CAGGTTACTATAGCAGAGATATTACTAATTATTAT-3'
F3-2	5'-ACTTTCAGGTTACTATAGCAGAGATATTACTAATT-3'
		F3-3	5'-CGTTGACTTTCAGGTTACTATAGCAGAGATATTAC-3'
F3-4	5'-CATCTCGTTGACTTTCAGGTTACTATAGCAGAGAT-3'
		F3-5	5'-TGTTTCATCTCGTTGACTTTCAGGTTACTATAGCA-3'
F3-6	5'-ACAGATGTTTCATCTCGTTGACTTTCAGGTTACTA-3'
		F3-7	5'-TGACAACAGATGTTTCATCTCGTTGACTTTCAGGT-3'
F3-8	5'-GTAAGTGACAACAGATGTTTCATCTCGTTGACTTT-3'
		F3-9	5'-GTACAGTAAGTGACAACAGATGTTTCATCTCGTTG-3'
F3-10	5'-TGCTTGTACAGTAAGTGACAACAGATGTTTCATCT-3'
		R3-1	5'-TGACTTAGATAAATTTTTAATTATGAGGTTTATGA-3'
R3-2	5'-GTTAGTGACTTAGATAAATTTTTAATTATGAGGTT-3'
		R3-3	5'-TCTCAGTTAGTGACTTAGATAAATTTTTAATTATG-3'
R3-4	5'-TTTATTCTCAGTTAGTGACTTAGATAAATTTTTAA-3'
		R3-5	5'-GAATATTTATTCTCAGTTAGTGACTTAGATAAATT-3'
R3-6	5'-ATTGAGAATATTTATTCTCAGTTAGTGACTTAGAT-3'
		R3-7	5'-ATCTAATTGAGAATATTTATTCTCAGTTAGTGACT-3'
R3-8	5'-TCTTCATCTAATTGAGAATATTTATTCTCAGTTAG-3'
		R3-9	5'-GTTGCTCTTCATCTAATTGAGAATATTTATTCTCA-3'
R3-10	5'-CATTGGTTGCTCTTCATCTAATTGAGAATATTTAT-3'

Probe 5	5'-TTGACTTCTATTTGTGCTTTTTAGCCTTTC/i6FAMdT//idSp/C/iBHQ1dT/ATTCCTTGTTTTAAT-3'
		F5-1	5'-GACAGAATGATTGAACTTTCATTAATTGACTTCTA-3'
F5-2	5'-AGAAAGACAGAATGATTGAACTTTCATTAATTGAC-3'
		F5-3	5'-TCAAAAGAAAGACAGAATGATTGAACTTTCATTAA-3'
F5-4	5'-CACACTCAAAAGAAAGACAGAATGATTGAACTTTC-3'
		F5-5	5'-TGCTTCACACTCAAAAGAAAGACAGAATGATTGAA-3'
F5-6	5'-CACTTTGCTTCACACTCAAAAGAAAGACAGAATGA-3'
		F5-7	5'-TATAACACTTTGCTTCACACTCAAAAGAAAGACAG-3'
F5-8	5'-GTGTTTATAACACTTTGCTTCACACTCAAAAGAAA-3'
		F5-9	5'-CAATAGTGTTTATAACACTTTGCTTCACACTCAAA-3'
F5-10	5'-TGCGGCAATAGTGTTTATAACACTTTGCTTCACAC-3'
		R5-1	5'-GCAGTTCAAGTGAGAACCAAAAGATAATAAGCATA-3'
R5-2	5'-ATCTTGCAGTTCAAGTGAGAACCAAAAGATAATAA-3'
		R5-3	5'-TTATGATCTTGCAGTTCAAGTGAGAACCAAAAGAT-3'
R5-4	5'-TTTCATTATGATCTTGCAGTTCAAGTGAGAACCAA-3'
		R5-5	5'-ACAAGTTTCATTATGATCTTGCAGTTCAAGTGAGA-3'
R5-6	5'-GCGTGACAAGTTTCATTATGATCTTGCAGTTCAAG-3'
		R5-7	5'-TTTAGGCGTGACAAGTTTCATTATGATCTTGCAGT-3'
R5-8	5'-GTTCGTTTAGGCGTGACAAGTTTCATTATGATCTT-3'
		R5-9	5'-TTCATGTTCGTTTAGGCGTGACAAGTTTCATTATG-3'
R5-10	5'-GAAATTTCATGTTCGTTTAGGCGTGACAAGTTTCA-3'

Probe 7	5'-TAAAATGAAAGATCTCAGTCCAAGATGGTA/i6FAMdT//idSp//iBHQ1dT/CTACTACCTAGGAAC-3'
		F7-1	5'-CCAGACGAATTCGTGGTGGTGACGGTAAAATGAAA-3'
F7-2	5'-AGCTACCAGACGAATTCGTGGTGGTGACGGTAAAA-3'
		F7-3	5'-CGAAGAGCTACCAGACGAATTCGTGGTGGTGACGG-3'
F7-4	5'-ACTACCGAAGAGCTACCAGACGAATTCGTGGTGGT-3'
		F7-5	5'-TGGCTACTACCGAAGAGCTACCAGACGAATTCGTG-3'
F7-6	5'-CAAATTGGCTACTACCGAAGAGCTACCAGACGAAT-3'
		F7-7	5'-ATGACCAAATTGGCTACTACCGAAGAGCTACCAGA-3'
F7-8	5'-TCCAGATGACCAAATTGGCTACTACCGAAGAGCTA-3'
		F7-9	5'-AGCAGTCCAGATGACCAAATTGGCTACTACCGAAG-3'
F7-10	5'-CCAATAGCAGTCCAGATGACCAAATTGGCTACTAC-3'
		R7-1	5'-TGTTAGCACCATAGGGAAGTCCAGCTTCTGGCCCA-3'
R7-2	5'-GTCTTTGTTAGCACCATAGGGAAGTCCAGCTTCTG-3'
		R7-3	5'-ATGCCGTCTTTGTTAGCACCATAGGGAAGTCCAGC-3'
R7-4	5'-ATATGATGCCGTCTTTGTTAGCACCATAGGGAAGT-3'
		R7-5	5'-AACCCATATGATGCCGTCTTTGTTAGCACCATAGG-3'
R7-6	5'-GTTGCAACCCATATGATGCCGTCTTTGTTAGCACC-3'
		R7-7	5'-CCTCAGTTGCAACCCATATGATGCCGTCTTTGTTA-3'
R7-8	5'-GGCTCCCTCAGTTGCAACCCATATGATGCCGTCTT-3'
		R7-9	5'-TTCAAGGCTCCCTCAGTTGCAACCCATATGATGCC-3'
R7-10	5'-GTGTATTCAAGGCTCCCTCAGTTGCAACCCATATG-3'

2. Screening of primers

Considering the importance of primers in RPA amplification, 10 pairs of primers were designed at both ends of one probe to select the most efficient primers and probes. In the primer pair screening, the first forward primer (F) is paired with the first to tenth reverse primers (R), respectively. Then, the reverse primers are screened again, and the first reverse primer (R) is paired with the first to tenth forward primers (F), respectively. Through two rounds of primer screening, the best primer pair with the highest slope and the shortest reaction time was found. And using the purified plasmid DNA containing SARS-CoV-2 partial gene sequence at 1X 10⁵Primers and probes were screened for specificity and sensitivity under copy. Finally, 16 pairs of primers were screened, and each gene fragment had two pairs of primers, one outer primer and one inner primer (see Table 2 below).

TABLE 2 efficient probes and primer sequences screened against 8 gene fragments (Frag.1-Frag.8) of SARS-CoV-2

Plasmid DNA (1X 10) containing SARS-CoV-2 partial sequence¹⁰copies/ul) was diluted sequentially to 1X 10 by 10-fold serial dilution⁹copies/ul,1×10⁸copies/ul,1×10⁷copies/ul,1×10⁶copies/ul,1×10⁵copies/ul,1×10⁵ copies/ul,1×10⁵copies/ul,1×10⁴copies/ul,1×10³copies/ul,5×10²copies/ul,3×10²copie/ul s, 2×10²copies/ul,1×10²copies/ul,1×10¹copies/ul. Using the RPA method, at the end of the 30-minute reaction, judging whether the difference of the fluorescence between the detected sample and the blank control has statistical significance, and judging that the standard is p<0.05 is positive, p>0.05 was negative. The results showed 16 pairs of primers and 8 stripsThe lowest limit of detection (LOD) of the probes was very different (see FIG. 2), indicating that primers and probes for different gene regions of the same viral sequence had different amplification efficiencies. Therefore, the primer pair and probe for Fragment 5 and Fragment 7 with the lowest LOD were selected for the nested RPA detection assay.

Example 2 detection of SARS-CoV-2 nucleic acid by nested RPA (nestRPA)

1. Method of producing a composite material

1.1 construction of SARS-CoV-2 Positive plasmid

3 target fragments (SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51) were synthesized, and BamHI and SalI restriction sites were added to both ends of the fragments. Each target fragment was mixed with T4 DNA ligase and pUC57 plasmid and ligated at 16 ℃ for 12 hours. The recombinant plasmid containing the target fragment is transformed into a recipient bacterium TOP10, and positive clones are screened by Luria Broth medium. The positive clones were verified to be correct using Sanger sequencing technology.

1.2 RPA reaction

The reaction was carried out in 50. mu.l of RPA reaction system using RAA nucleic acid amplification kit (QITIAN Bio Co., Wuxi, China). The reaction mixture contained the following ingredients: mu.l of hydration buffer, 2.1. mu.l of forward primer (10. mu.M), 2.1. mu.l of reverse primer (10. mu.M), 0.6. mu.l of probe (10. mu.M), 1. mu.l of DNA template, 16.7. mu.l of purified water and 2.5. mu.l of 280nM magnesium acetate solution (MgAc). The reaction mixture is added to a reaction tube containing lyophilized reagent powder comprising the recombinase, the single-strand binding protein and the DNA polymerase. The reaction tubes were incubated in a fluorescence isothermal amplification apparatus (QITIAN Bio Co., Wuxi, China) for amplification at 39 ℃ for 30 minutes. After amplification is complete, the RPA amplification product is purified.

1.3 nested RPA detection

In order to develop a detection technology with ultrahigh sensitivity, the invention provides a nested RPA detection method (nestRPA) based on the RPA technology, and the nested RPA detection method is applied to SARS-CoV-2 nucleic acid detection. The rationale for the nestRPA is: a first segment of the target gene is amplified using the outer primers, and then a second segment of the target gene, which is completely within the first amplified segment, is amplified using the inner primers. In order to eliminate the effect of the enzyme's fluorescent signal, no fluorescent probe was included in the first RPA reaction system. In the second RPA reaction, a fluorescent probe was added. The specific procedure for nested RPA using the primer pairs and probes of Fragment 5 and Fragment 7 (see Table 3) is as follows.

Nested RPA detection involves two rounds of reaction. In the first reaction, the reaction mixture comprises the following components: 25 μ l of hydration buffer, 2.1 μ l of external forward primer (10 μ M), 2.1 μ l of external reverse primer (10 μ M), 17.7 μ l of DNA template (plasmid DNA or nucleic acid extract of clinical specimen), 2.5 μ l of magnesium acetate solution (MgAc, 280 nM). The RPA reaction mixture is added to the reaction tube containing the lyophilized reagent powder. The first round of RPA reaction was incubated in the isothermal amplification apparatus at 39 ℃ for 20 minutes. All the products of the first round of reaction are then used as templates for the second round of reaction. In the second reaction, the first reaction product is mixed with 25.2. mu.l of hydration buffer, 2.1. mu.l of internal forward primer (10. mu.M), 2.1. mu.l of internal reverse primer (10. mu.M) and 0.6. mu.L of probe (10. mu.M), and then the mixture is added to a new reaction tube containing lyophilized reagent powder, mixed and centrifuged. The second RPA reaction was incubated in a fluorescence-isothermal amplification instrument (QITIAN Bio co., Wuxi, China) at 39 ℃ for 30 minutes, the results of the nestRPA fluorescence signal were recorded in the FAM observation channel, and the fluorescence value of the positive results exceeded the background fluorescence signal index multiple (p < 0.05).

1.4 statistical analysis

Statistical analysis was performed using SPSS 19.0 software (Chicago, IL, USA). And simultaneously evaluating the experimental result by using independent sample t test and chi-square test. All data analyses, p <0.05 represents significant differences.

2. Results

The positive plasmid containing SARS-CoV-2DNA of 1copy/ul can be detected by nested RPA detection using the primer pair and probe of Fragment 5 and Fragment 7 selected (see Table 3 and FIG. 3), which shows that the nested RPA detection of SARS-CoV-2 nucleic acid has ultrahigh sensitivity.

TABLE 3 nest type RPA test result statistics of low concentration samples

Example 3 comparison of results of clinical samples tested by the NestRPA and qPCR methods

1. Method of producing a composite material

1.1 clinical samples

All cases were examined by real-time quantitative PCR (qPCR) to determine whether or not SARS-CoV-2 was infected. 189 samples (nasopharyngeal swabs or sputum) from 139 patients were collected in Shenzhen third people hospital during the period from 2/1/2020 to 3/24/2020. 20 samples from 17 patients with positive qPCR test results (6 samples with positive qPCR test results from positive patients), 101 samples from 73 patients with negative qPCR test results, 42 samples from 20 patients with multiple hospitalizations with positive qPCR test results (6 of the qPCR test results are positive, 36 of the qPCR test results are negative), and 32 samples from 32 patients with repeated work.

1.2 Total nucleic acid extraction of samples

According to the procedure of the nucleic acid extraction kit (Huayin Biotech co., Shenzhen, China), a reaction solution was prepared, and total nucleic acids including DNA and RNA of a clinical sample were extracted using an automatic nucleic acid extractor.

1.3 fluorescent quantitative PCR (qPCR) assay

Performing reverse transcription and qPCR reaction in the same reaction tube by using one-step method qRT-PCR kit (

II U + One Step qRT-PCR Probe kit, Vazyme Biotech Co., Ltd.). The reaction system was 20. mu.l containing 2 Xone Step Q Probe Mix, One Step Q Probe Enzyme Mix,50 XROX Reference Dye 1, forward and reverse Gene Specific Primers (GSP) (10. mu.M), TaqMan Probe (10. mu.M) and RNA template. ORF1a/b gene detection requires the use of ORF1ab-F, ORF1ab-R and ORF1 ab-P. N-F, N-R and N-P are required for detecting the N gene. Primer and probe sequences were published on the CDC website (http:// ivdc. china CDC. cn/kyjz/202001/t20200121_211337. html). Mixing the reactionAnd placing the mixture in a qPCR detector to perform one-step RT-qPCR reaction. Thermal cycling reaction parameters are described in the literature [ Corman VM, Landt O, Kaiser M, Molenkamp R, Meijer A, Chu DKW, Bleicker T, Bruinink S, Schneider J, Schmidt ML, Mulders DGJC, Haagmans BL, van der Veer B, van den Brink S, Wijsman L, Goderski G, Romette JL, Ellis J, Zambon M, Peiris M, Goosens H, Reusken C, Koopmans MPG, Drosen C.detection of 2019 novel coronavirs (2019-nCoV) by real RT-PCR. Surveill. Jan; 25(3).]。

1.4 detection of RNA from clinical specimens using the nested RPA detection method described in example 2 (nestRPA). And (3) detecting the sample with positive qPCR detection result by using the method of the invention for the nestRPA, and detecting 1 case of the positive qPCR detection sample by using the method for the nestRPA after continuously diluting the positive qPCR detection sample by 11 times and 10 times. Samples with negative qPCR detection results were also tested by the nestRPA method of the present invention.

2. Results

2.1 nasal swabs or sputum samples (6 samples from positive patients) were collected from 17 COVID-19 patients, all of which were positive for the detection of SARS-CoV-2 nucleic acid by the qPCR method. The detection by the method of the invention for the nestRPA shows that the consistency rate of the detection result of the method of the invention for the nucleic acid positive sample and the detection result of the qPCR method is 100 percent (20/20).

2.2 one of the positive samples was selected and the sensitivity of the inventive nestRPA method was tested by 10-fold serial dilution. The results showed that positive results were still detected by the nested RPA method of the present invention (nestRPA) after 10-fold serial dilution of SARS-CoV-2 nucleic acid positive samples for 11 times, whereas the results of qPCR for 3 rd diluted samples were negative (Table 4).

TABLE 4 comparison of the results of nested RPA and qPCR assays on a positive sample after 10-fold serial dilution

ND means no detection.

2.3 collecting 101 nasal swab or sputum samples from 73 COVID-19 patients or other disease patients, all of which were negative for detecting SARS-CoV-2 nucleic acid by qPCR method, the nested RPA method of the present invention gave a positive result of 32.67% (33/101) (Table 5), indicating that there was false negative for detecting SARS-CoV-2 nucleic acid in clinical COVID-19 patients by qPCR method.

TABLE 5 detection results of nested RPA on qPCR-negative samples

ND means that the sample size is insufficient and no detection is performed.

2.4 to dynamically observe the changes in SARS-CoV-2 nucleic acid in patients with repeated hospitalizations of COVID-19, 42 samples from 20 COVID-19 patients with multiple positive were collected for SARS-CoV-2 nucleic acid detection. Wherein, 6 samples with positive qPCR detection (included in 20 positive samples with positive qPCR detection in the example 2.1) are also positive by the nested RPA detection result of the invention; 36 samples with negative qPCR detection results, 91.67% (33/36) of nucleic acid detection results are positive (table 6) by using the nested RPA detection method, and the detection results are basically consistent with clinical diagnosis results, which shows that the detection method with ultrahigh sensitivity can be used for diagnosing patients suspected of having new coronary pneumonia clinically.

TABLE 6 comparison of the results of nested RPA and qPCR for COVID-19 patients with a relapse

2.5 to investigate whether asymptomatic SARS-CoV-2-nucleic acid-positive patients were present in healthy populations, 32 throat swab samples from repeat workers were collected, all of which were negative for SARS-CoV-2 nucleic acid detection by qPCR, whereas 12.5% (4/32) of the samples obtained by the nested RPA method of the present invention were positive (see FIGS. 4 and 7), indicating that the ultra-high sensitivity nucleic acid detection method of the present invention is of great importance for early detection of asymptomatic SARS-CoV-2-bearing patients.

TABLE 7 nest type RPA detection result of the repeat worker physical examination sample

2.6 in order to avoid false positives in the nested RPA assay of the present invention, the amplification products of all the samples tested positive for nested RPA in examples 2.1-2.5 were subjected to high throughput sequencing, which showed 100% of the detected fragments as target sequences (FIG. 5).

In summary, as shown in Table 8 below, 189 clinical samples from 139 patients (107 patients with COVID-19 confirmed diagnosis) were tested in the present invention, wherein the results of qPCR testing were 20 positive samples and 169 negative samples. 90 positive samples and 99 negative samples are detected by the nested RPA of the invention, wherein the detection result of 119 samples is consistent with the qPCR detection result (comprising 20 positive samples and 99 negative samples), the qPCR detection result is positive 20 samples, the nested RPA detection result of the invention is also all positive, and in 169 samples with the qPCR detection result being negative, 37.04% (70/169) positive results are obtained by the nested RPA of the invention, and statistical analysis shows that the nested RPA detection result with ultrahigh sensitivity of the invention is obviously different from the qPCR detection result (P is 0.0000).

TABLE 8 statistical Table of results of SARS-CoV-2 nucleic acid detection by nested RPA and qPCR, respectively

The above-mentioned embodiments only express the embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Sequence listing

<110> Huangwanqiu

Huang Jian

<120> primer pair, probe and kit for detecting SARS-CoV-2 by using nested RPA technology and application thereof

<160> 51

<170> PatentIn version 3.3

<210> 1

<211> 178

<212> DNA

<213> SARS-CoV-2 novel coronavirus (Severe acid respiratory syndrome coronavirus 2)

<400> 1

tttgaccgtt attttaaata ttgggatcag acataccacc caaattgtgt taactgtttg 60

gatgacagat gcattctgca ttgtgcaaac tttaatgttt tattctctac agtgttccca 120

cctacaagtt ttggaccact agtgagaaaa atatttgttg atggtgttcc atttgtag 178

<210> 2

<211> 178

<212> DNA

<400> 2

caaacctttt gagagagata tttcaactga aatctatcag gccggtagca caccttgtaa 60

tggtgttgaa ggttttaatt gttactttcc tttacaatca tatggtttcc aacccactaa 120

tggtgttggt taccaaccat acagagtagt agtactttct tttgaacttc tacatgca 178

<210> 3

<211> 168

<212> DNA

<400> 3

catctcgttg actttcaggt tactatagca gagatattac taattattat gaggactttt 60

aaagtttcca tttggaatct tgattacatc ataaacctca taattaaaaa tttatctaag 120

tcactaactg agaataaata ttctcaatta gatgaagagc aaccaatg 168

<210> 4

<211> 133

<212> DNA

<400> 4

atacgagggc aattcaccat ttcatcctct agctgataac aaatttgcac tgacttgctt 60

tagcactcaa tttgcttttg cttgtcctga cggcgtaaaa cacgtctatc agttacgtgc 120

cagatcagtt tca 133

<210> 5

<211> 199

<212> DNA

<400> 5

tgcggcaata gtgtttataa cactttgctt cacactcaaa agaaagacag aatgattgaa 60

ctttcattaa ttgacttcta tttgtgcttt ttagcctttc tgctattcct tgttttaatt 120

atgcttatta tcttttggtt ctcacttgaa ctgcaagatc ataatgaaac ttgtcacgcc 180

taaacgaaca tgaaatttc 199

<210> 6

<211> 201

<212> DNA

<400> 6

agtttcctgt ttacctttta caattaattg ccaggaacct aaattgggta gtcttgtagt 60

gcgttgttcg ttctatgaag actttttaga gtatcatgac gttcgtgttg ttttagattt 120

catctaaacg aacaaactaa aatgtctgat aatggacccc aaaatcagcg aaatgcaccc 180

cgcattacgt ttggtggacc c 201

<210> 7

<211> 163

<212> DNA

<400> 7

caaattggct actaccgaag agctaccaga cgaattcgtg gtggtgacgg taaaatgaaa 60

gatctcagtc caagatggta tttctactac ctaggaactg ggccagaagc tggacttccc 120

tatggtgcta acaaagacgg catcatatgg gttgcaactg agg 163

<210> 8

<211> 174

<212> DNA

<400> 8

tcatcacgta gtcgcaacag ttcaagaaat tcaactccag gcagcagtag gggaacttct 60

cctgctagaa tggctggcaa tggcggtgat gctgctcttg ctttgctgct gcttgacaga 120

ttgaaccagc ttgagagcaa aatgtctggt aaaggccaac aacaacaagg ccaa 174

<210> 9

<211> 48

<212> DNA

<213> Artificial sequence (Artificial)

<220>

<221> misc_feature

<222> (31)..(31)

<223> n is i6FAMdT

<220>

<221> misc_feature

<222> (32)..(32)

<223> n is idSp

<220>

<221> misc_feature

<222> (33)..(33)

<223> n is iBHQ1dT

<400> 9

gatgacagat gcattctgca ttgtgcaaac nnnaatgttt tattctct 48

<210> 10

<211> 35

<212> DNA

<213> Artificial sequence (Artificial)

<400> 10

tttgaccgtt attttaaata ttgggatcag acata 35

<210> 11

<211> 35

<212> DNA

<213> Artificial sequence (Artificial)

<400> 11

ctacaaatgg aacaccatca acaaatattt ttctc 35

<210> 12

<211> 35

<212> DNA

<213> Artificial sequence (Artificial)

<400> 12

atcagacata ccacccaaat tgtgttaact gtttg 35

<210> 13

<211> 35

<212> DNA

<213> Artificial sequence (Artificial)

<400> 13

catcaacaaa tatttttctc actagtggtc caaaa 35

<210> 14

<211> 48

<212> DNA

<213> Artificial sequence (Artificial)

<220>

<221> misc_feature

<222> (31)..(31)

<223> n is i6FAMdT

<220>

<221> misc_feature

<222> (32)..(32)

<223> n is idSp

<220>

<221> misc_feature

<222> (33)..(33)

<223> n is iBHQ1dT

<400> 14

tgtaatggtg ttgaaggttt taattgttac nnncctttac aatcatat 48

<210> 15

<211> 35

<212> DNA

<213> Artificial sequence (Artificial)

<400> 15

caaacctttt gagagagata tttcaactga aatct 35

<210> 16

<211> 35

<212> DNA

<213> Artificial sequence (Artificial)

<400> 16

tgcatgtaga agttcaaaag aaagtactac tactc 35

<210> 17

<211> 35

<212> DNA

<213> Artificial sequence (Artificial)

<400> 17

cttttgagag agatatttca actgaaatct atcag 35

<210> 18

<211> 35

<212> DNA

<213> Artificial sequence (Artificial)

<400> 18

agttcaaaag aaagtactac tactctgtat ggttg 35

<210> 19

<211> 48

<212> DNA

<213> Artificial sequence (Artificial)

<220>

<221> misc_feature

<222> (31)..(31)

<223> n is i6FAMdT

<220>

<221> misc_feature

<222> (32)..(32)

<223> n is idSp

<220>

<221> misc_feature

<222> (33)..(33)

<223> n is iBHQ1dT

<400> 19

taattattat gaggactttt aaagtttcca nnnggaatct tgattaca 48

<210> 20

<211> 35

<212> DNA

<213> Artificial sequence (Artificial)

<400> 20

catctcgttg actttcaggt tactatagca gagat 35

<210> 21

<211> 35

<212> DNA

<213> Artificial sequence (Artificial)

<400> 21

cattggttgc tcttcatcta attgagaata tttat 35

<210> 22

<211> 35

<212> DNA

<213> Artificial sequence (Artificial)

<400> 22

actttcaggt tactatagca gagatattac taatt 35

<210> 23

<211> 35

<212> DNA

<213> Artificial sequence (Artificial)

<400> 23

gttagtgact tagataaatt tttaattatg aggtt 35

<210> 24

<211> 48

<212> DNA

<213> Artificial sequence (Artificial)

<220>

<221> misc_feature

<222> (31)..(31)

<223> n is i6FAMdT

<220>

<221> misc_feature

<222> (32)..(32)

<223> n is idSp

<220>

<221> misc_feature

<222> (33)..(33)

<223> n is iBHQ1dT

<400> 24

aaatttgcac tgacttgctt tagcactcaa nnngcttttg cttgtcct 48

<210> 25

<211> 35

<212> DNA

<213> Artificial sequence (Artificial)

<400> 25

atacgagggc aattcaccat ttcatcctct agctg 35

<210> 26

<211> 35

<212> DNA

<213> Artificial sequence (Artificial)

<400> 26

tgaaactgat ctggcacgta actgatagac gtgtt 35

<210> 27

<211> 35

<212> DNA

<213> Artificial sequence (Artificial)

<400> 27

accatttcat cctctagctg ataacaaatt tgcac 35

<210> 28

<211> 35

<212> DNA

<213> Artificial sequence (Artificial)

<400> 28

ctgatctggc acgtaactga tagacgtgtt ttacg 35

<210> 29

<211> 49

<212> DNA

<213> Artificial sequence (Artificial)

<220>

<221> misc_feature

<222> (31)..(31)

<223> n is i6FAMdT

<220>

<221> misc_feature

<222> (32)..(32)

<223> n is idSp

<220>

<221> misc_feature

<222> (34)..(34)

<223> n is iBHQ1dT

<400> 29

ttgacttcta tttgtgcttt ttagcctttc nncnattcct tgttttaat 49

<210> 30

<211> 35

<212> DNA

<213> Artificial sequence (Artificial)

<400> 30

tgcggcaata gtgtttataa cactttgctt cacac 35

<210> 31

<211> 35

<212> DNA

<213> Artificial sequence (Artificial)

<400> 31

gaaatttcat gttcgtttag gcgtgacaag tttca 35

<210> 32

<211> 35

<212> DNA

<213> Artificial sequence (Artificial)

<400> 32

gacagaatga ttgaactttc attaattgac ttcta 35

<210> 33

<211> 35

<212> DNA

<213> Artificial sequence (Artificial)

<400> 33

gcagttcaag tgagaaccaa aagataataa gcata 35

<210> 34

<211> 51

<212> DNA

<213> Artificial sequence (Artificial)

<220>

<221> misc_feature

<222> (33)..(33)

<223> n is i6FAMdT

<220>

<221> misc_feature

<222> (35)..(35)

<223> n is idSp

<220>

<221> misc_feature

<222> (36)..(36)

<223> n is iBHQ1dT

<400> 34

ttctatgaag actttttaga gtatcatgac gtncnngttg ttttagattt c 51

<210> 35

<211> 35

<212> DNA

<213> Artificial sequence (Artificial)

<400> 35

agtttcctgt ttacctttta caattaattg ccagg 35

<210> 36

<211> 35

<212> DNA

<213> Artificial sequence (Artificial)

<400> 36

agcgaaatgc accccgcatt acgtttggtg gaccc 35

<210> 37

<211> 35

<212> DNA

<213> Artificial sequence (Artificial)

<400> 37

ccaggaacct aaattgggta gtcttgtagt gcgtt 35

<210> 38

<211> 35

<212> DNA

<213> Artificial sequence (Artificial)

<400> 38

gattttgggg tccattatca gacattttag tttgt 35

<210> 39

<211> 48

<212> DNA

<213> Artificial sequence (Artificial)

<220>

<221> misc_feature

<222> (31)..(31)

<223> n is i6FAMdT

<220>

<221> misc_feature

<222> (32)..(32)

<223> n is idSp

<220>

<221> misc_feature

<222> (33)..(33)

<223> n is iBHQ1dT

<400> 39

taaaatgaaa gatctcagtc caagatggta nnnctactac ctaggaac 48

<210> 40

<211> 35

<212> DNA

<213> Artificial sequence (Artificial)

<400> 40

caaattggct actaccgaag agctaccaga cgaat 35

<210> 41

<211> 35

<212> DNA

<213> Artificial sequence (Artificial)

<400> 41

cctcagttgc aacccatatg atgccgtctt tgtta 35

<210> 42

<211> 35

<212> DNA

<213> Artificial sequence (Artificial)

<400> 42

ccagacgaat tcgtggtggt gacggtaaaa tgaaa 35

<210> 43

<211> 35

<212> DNA

<213> Artificial sequence (Artificial)

<400> 43

gtctttgtta gcaccatagg gaagtccagc ttctg 35

<210> 44

<211> 49

<212> DNA

<213> Artificial sequence (Artificial)

<220>

<221> misc_feature

<222> (32)..(32)

<223> n is i6FAMdT

<220>

<221> misc_feature

<222> (33)..(33)

<223> n is idSp

<220>

<221> misc_feature

<222> (34)..(34)

<223> n is iBHQ1dT

<400> 44

tggctggcaa tggcggtgat gctgctcttg cnnngctgct gcttgacag 49

<210> 45

<211> 35

<212> DNA

<213> Artificial sequence (Artificial)

<400> 45

tcatcacgta gtcgcaacag ttcaagaaat tcaac 35

<210> 46

<211> 35

<212> DNA

<213> Artificial sequence (Artificial)

<400> 46

ttggccttgt tgttgttggc ctttaccaga cattt 35

<210> 47

<211> 35

<212> DNA

<213> Artificial sequence (Artificial)

<400> 47

gaaattcaac tccaggcagc agtaggggaa cttct 35

<210> 48

<211> 35

<212> DNA

<213> Artificial sequence (Artificial)

<400> 48

ttggccttta ccagacattt tgctctcaag ctggt 35

<210> 49

<211> 2139

<212> DNA

<213> Artificial sequence (Artificial)

<400> 49

tttcttatta caaattggga gcttcgcagc gtgtagcagg tgactcaggt tttgctgcat 60

acagtcgcta caggattggc aactataaat taaacacaga ccattccagt agcagtgaca 120

atattgcttt gcttgtacag taagtgacaa cagatgtttc atctcgttga ctttcaggtt 180

actatagcag agatattact aattattatg aggactttta aagtttccat ttggaatctt 240

gattacatca taaacctcat aattaaaaat ttatctaagt cactaactga gaataaatat 300

tctcaattag atgaagagca accaatggag attgattaaa cgaacatgaa aattattctt 360

ttcttggcac tgataacact cgctacttgt gagctttatc actaccaaga gtgtgttaga 420

ggtacaacag tacttttaaa agaaccttgc tcttctggaa catacgaggg caattcacca 480

tttcatcctc tagctgataa caaatttgca ctgacttgct ttagcactca atttgctttt 540

gcttgtcctg acggcgtaaa acacgtctat cagttacgtg ccagatcagt ttcacctaaa 600

ctgttcatca gacaagagga agttcaagaa ctttactctc caatttttct tattgttgcg 660

gcaatagtgt ttataacact ttgcttcaca ctcaaaagaa agacagaatg attgaacttt 720

cattaattga cttctatttg tgctttttag cctttctgct attccttgtt ttaattatgc 780

ttattatctt ttggttctca cttgaactgc aagatcataa tgaaacttgt cacgcctaaa 840

cgaacatgaa atttcttgtt ttcttaggaa tcatcacaac tgtagctgca tttcaccaag 900

aatgtagttt acagtcatgt actcaacatc aaccatatgt agttgatgac ccgtgtccta 960

ttcacttcta ttctaaatgg tatattagag taggagctag aaaatcagca cctttaattg 1020

aattgtgcgt ggatgaggct ggttctaaat cacccattca gtacatcgat atcggtaatt 1080

atacagtttc ctgtttacct tttacaatta attgccagga acctaaattg ggtagtcttg 1140

tagtgcgttg ttcgttctat gaagactttt tagagtatca tgacgttcgt gttgttttag 1200

atttcatcta aacgaacaaa ctaaaatgtc tgataatgga ccccaaaatc agcgaaatgc 1260

accccgcatt acgtttggtg gaccctcaga ttcaactggc agtaaccaga atggagaacg 1320

cagtggggcg cgatcaaaac aacgtcggcc ccaaggttta cccaataata ctgcgtcttg 1380

gttcaccgct ctcactcaac atggcaagga agaccttaaa ttccctcgag gacaaggcgt 1440

tccaattaac accaatagca gtccagatga ccaaattggc tactaccgaa gagctaccag 1500

acgaattcgt ggtggtgacg gtaaaatgaa agatctcagt ccaagatggt atttctacta 1560

cctaggaact gggccagaag ctggacttcc ctatggtgct aacaaagacg gcatcatatg 1620

ggttgcaact gagggagcct tgaatacacc aaaagatcac attggcaccc gcaatcctgc 1680

taacaatgct gcaatcgtgc tacaacttcc tcaaggaaca acattgccaa aaggcttcta 1740

cgcagaaggg agcagaggcg gcagtcaagc ctcttctcgt tcctcatcac gtagtcgcaa 1800

cagttcaaga aattcaactc caggcagcag taggggaact tctcctgcta gaatggctgg 1860

caatggcggt gatgctgctc ttgctttgct gctgcttgac agattgaacc agcttgagag 1920

caaaatgtct ggtaaaggcc aacaacaaca aggccaaact gtcactaaga aatctgctgc 1980

tgaggcttct aagaagcctc ggcaaaaacg tactgccact aaagcataca atgtaacaca 2040

agctttcggc agacgtggtc cagaacaaac ccaaggaaat tttggggacc aggaactaat 2100

cagacaagga actgattaca aacattggcc gcaaattgc 2139

<210> 50

<211> 1587

<212> DNA

<213> Artificial sequence (Artificial)

<400> 50

aaacatgact tctttaagtt tagaatagac ggtgacatgg taccacatat atcacgtcaa 60

cgtcttacta aatacacaat ggcagacctc gtctatgctt taaggcattt tgatgaaggt 120

aattgtgaca cattaaaaga aatacttgtc acatacaatt gttgtgatga tgattatttc 180

aataaaaagg actggtatga ttttgtagaa aacccagata tattacgcgt atacgccaac 240

ttaggtgaac gtgtacgcca agctttgtta aaaacagtac aattctgtga tgccatgcga 300

aatgctggta ttgttggtgt actgacatta gataatcaag atctcaatgg taactggtat 360

gatttcggtg atttcataca aaccacgcca ggtagtggag ttcctgttgt agattcttat 420

tattcattgt taatgcctat attaaccttg accagggctt taactgcaga gtcacatgtt 480

gacactgact taacaaagcc ttacattaag tgggatttgt taaaatatga cttcacggaa 540

gagaggttaa aactctttga ccgttatttt aaatattggg atcagacata ccacccaaat 600

tgtgttaact gtttggatga cagatgcatt ctgcattgtg caaactttaa tgttttattc 660

tctacagtgt tcccacctac aagttttgga ccactagtga gaaaaatatt tgttgatggt 720

gttccatttg tagtttcaac tggataccac ttcagagagc taggtgttgt acataatcag 780

gatgtaaact tacatagctc tagacttagt tttaaggaat tacttgtgta tgctgctgac 840

cctgctatgc acgctgcttc tggtaatcta ttactagata aacgcactac gtgcttttca 900

gtagctgcac ttactaacaa tgttgctttt caaactgtca aacccggtaa ttttaacaaa 960

gacttctatg actttgctgt gtctaagggt ttctttaagg aaggaagttc tgttgaatta 1020

aaacacttct tctttgctca ggatggtaat gctgctatca gcgattatga ctactatcgt 1080

tataatctac caacaatgtg tgatatcaga caactactat ttgtagttga agttgttgat 1140

aagtactttg attgttacga tggtggctgt attaatgcta accaagtcat cgtcaacaac 1200

ctagacaaat cagctggttt tccatttaat aaatggggta aggctagact ttattatgat 1260

tcaatgagtt atgaggatca agatgcactt ttcgcatata caaaacgtaa tgtcatccct 1320

actataactc aaatgaatct taagtatgcc attagtgcaa agaatagagc tcgcaccgta 1380

gctggtgtct ctatctgtag tactatgacc aatagacagt ttcatcaaaa attattgaaa 1440

tcaatagccg ccactagagg agctactgta gtaattggaa caagcaaatt ctatggtggt 1500

tggcacaaca tgttaaaaac tgtttatagt gatgtagaaa accctcacct tatgggttgg 1560

gattatccta aatgtgatag agccatg 1587

<210> 51

<211> 741

<212> DNA

<213> Artificial sequence (Artificial)

<400> 51

tcctatataa ttccgcatca ttttccactt ttaagtgtta tggagtgtct cctactaaat 60

taaatgatct ctgctttact aatgtctatg cagattcatt tgtaattaga ggtgatgaag 120

tcagacaaat cgctccaggg caaactggaa agattgctga ttataattat aaattaccag 180

atgattttac aggctgcgtt atagcttgga attctaacaa tcttgattct aaggttggtg 240

gtaattataa ttacctgtat agattgttta ggaagtctaa tctcaaacct tttgagagag 300

atatttcaac tgaaatctat caggccggta gcacaccttg taatggtgtt gaaggtttta 360

attgttactt tcctttacaa tcatatggtt tccaacccac taatggtgtt ggttaccaac 420

catacagagt agtagtactt tcttttgaac ttctacatgc accagcaact gtttgtggac 480

ctaaaaagtc tactaatttg gttaaaaaca aatgtgtcaa tttcaacttc aatggtttaa 540

caggcacagg tgttcttact gagtctaaca aaaagtttct gcctttccaa caatttggca 600

gagacattgc tgacactact gatgctgtcc gtgatccaca gacacttgag attcttgaca 660

ttacaccatg ttcttttggt ggtgtcagtg ttataacacc aggaacaaat acttctaacc 720

aggttgctgt tctttatcag g 741

Claims

1. A primer pair for detecting SARS-CoV-2 novel coronavirus by nested RPA, the primer pair is designed aiming at any one or more than two genes in the following SARS-CoV-2 novel coronavirus complete gene sequence: ORF1ab, S gene, ORF6, ORF7a, ORF7b, ORF8, N gene, ORF 10.

2. The primer pair of claim 1, wherein the primer pair is a primer pair for amplifying SEQ ID NO: 1 to SEQ ID NO: 8 in the sequence listing.

3. The primer pair of claim 2, wherein the amplification of the sequence of SEQ ID NO: 1 comprises the following components: the outer primer pair SEQ ID NO: 10 and SEQ ID NO: 11, inner primer pair SEQ ID NO: 12 and SEQ ID NO: 13.

4. the primer pair of claim 2, wherein the amplification of the sequence of SEQ ID NO: 2 comprises the following components: the outer primer pair SEQ ID NO: 15 and SEQ ID NO: 16, inner primer pair SEQ ID NO: 17 and SEQ ID NO: 18.

5. the primer pair of claim 2, wherein the amplification of the sequence of SEQ ID NO: 3 comprises the following components: the outer primer pair SEQ ID NO: 20 and SEQ ID NO: 21, inner primer pair SEQ ID NO: 22 and SEQ ID NO: 23.

6. the primer pair of claim 2, wherein the amplification of the sequence of SEQ ID NO: 4 comprises the following components: the outer primer pair SEQ ID NO: 25 and SEQ ID NO: 26, inner primer pair SEQ ID NO: 27 and SEQ ID NO: 28.

7. the primer pair of claim 2, wherein the amplification of the sequence of SEQ ID NO: 5 comprises the following components: the outer primer pair SEQ ID NO: 30 and SEQ ID NO: 31, inner primer pair SEQ ID NO: 32 and SEQ ID NO: 33.

8. the primer pair of claim 2, wherein the amplification of the sequence of SEQ ID NO: 6 comprises the following primer pairs: the outer primer pair SEQ ID NO: 35 and SEQ ID NO: 36, inner primer pair SEQ ID NO: 37 and SEQ ID NO: 38.

9. the primer pair of claim 2, wherein the amplification of the sequence of SEQ ID NO: 7 comprises the following primer pairs: the outer primer pair SEQ ID NO: 40 and SEQ ID NO: 41, inner primer pair SEQ ID NO: 42 and SEQ ID NO: 43.

10. the primer pair of claim 2, wherein the amplification of the sequence of SEQ ID NO: 8 comprises the following primer pairs: the outer primer pair SEQ ID NO: 45 and SEQ ID NO: 46, inner primer pair SEQ ID NO: 47 and SEQ ID NO: 48.

11. a probe for detecting SARS-CoV-2 new coronavirus by nested RPA, said probe is a probe specifically designed for any one or more than two genes in the following SARS-CoV-2 new coronavirus complete gene sequence: ORF1ab, S gene, ORF6, ORF7a, ORF7b, ORF8, N gene, ORF 10.

12. The probe of claim 11, wherein the probe hybridizes to SEQ ID NO: 1 to SEQ ID NO: 8 in the presence of a primer, and specifically hybridizing a part of the sequence of any one of the gene fragments.

13. The probe of claim 12, wherein the probe comprises:

and SEQ ID NO: 1 gene fragment having the sequence of SEQ ID NO: 9;

and SEQ ID NO: 2 gene fragment having the sequence of SEQ ID NO: 14;

and SEQ ID NO: 4 gene fragment having the sequence of SEQ ID NO: 24;

and SEQ ID NO: 5 gene fragment having the sequence of SEQ ID NO: 29;

and SEQ ID NO: 6 gene fragment having the sequence of SEQ ID NO: 34;

and SEQ ID NO: 7 gene fragment having the sequence of SEQ ID NO: 39;

14. A kit for detecting SARS-CoV-2 novel coronavirus, comprising the primer set of any one of claims 1 to 10, and the probe of any one of claims 11 to 13 corresponding to the primer set.

15. The kit of claim 14, wherein the kit comprises the primer pair of claim 7 and the primer of SEQ ID NO: 29, or a fragment thereof.

16. The kit of claim 14, wherein the kit comprises the primer pair of claim 9 and the primer of SEQ ID NO: 39, or a fragment thereof.

17. Use of a kit according to any one of claims 14 to 16 for the preparation of a product for the detection of the SARS-CoV-2 novel coronavirus.

18. A method for detecting viruses with nested RPAs, comprising the steps of:

19. A primer pair for detecting SARS-CoV-2 new coronavirus, the primer pair is designed aiming at any one or more than two genes in the following SARS-CoV-2 new coronavirus whole gene sequence: ORF1ab, S gene, ORF6, ORF7a, ORF7b, ORF8, N gene, ORF 10.

20. The primer pair of claim 19, wherein the primer pair is designed for three genes of SARS-CoV-2 coronavirus ORF7a, ORF7b and ORF8, and is used for amplifying the nucleotide sequence shown in SEQ ID NO: 5.

21. A probe for detecting SARS-CoV-2 new type coronavirus, said probe is designed for any one or more than two genes in the following SARS-CoV-2 new type coronavirus whole gene sequence: ORF1ab, S gene, ORF6, ORF7a, ORF7b, ORF8, N gene, ORF 10.

22. The probe of claim 21, wherein the probe hybridizes to SEQ ID NO: 5, and specifically hybridizing partial sequences of the gene segments shown in the specification.