CN111961763A - Novel gene chip for detecting coronavirus - Google Patents
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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- C12Q1/701—Specific hybridization probes
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6813—Hybridisation assays
- C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
- C12Q1/6837—Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips
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Abstract
The present invention provides a gene chip which can be used for detecting the existence of novel coronavirus SARS-CoV-2 and, if existing, determining the genome sequence of SARS-CoV-2; a kit comprising the gene chip; and a method for detecting the presence of the novel coronavirus SARS-CoV-2 using the gene chip and, if present, determining the genomic sequence of SARS-CoV-2.
Description
Technical Field
The present invention provides a gene chip which can be used for detecting the presence of the novel coronavirus SARS-CoV-2 and, if present, determining the genomic sequence of SARS-CoV-2.
Background
The severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) caused epidemic situation has spread globally, and as soon as 2020, 7, 14 days, more than 1300 million people have been diagnosed with the novel coronavirus pneumonia COVID-19 globally, more than 57 million patients die, and the number of people is still increasing rapidly. With the development of COVID-19 epidemic situation worldwide, a plurality of countries enter public health emergency states, and global scientists grasp and research better detection, treatment, prevention and control means. At present, SARS-CoV-2 virus can be detected by two major categories, antibody detection and nucleic acid detection.
The antibody is a kind of immunoglobulin which stimulates immune cells of the human body to produce specifically after the virus infects the human body, and the existence of the antibody can be detected through the antigen by utilizing the principle of combining the specificity of the antigen and the antibody, thereby indirectly proving that the human body is infected with the novel coronavirus. The antibody detection has the greatest advantages of convenience, rapidness and short detection time; however, antibodies may not be produced in human bodies in the early stage of infection of diseases, so that the antibody detection has a window stage and individual immune response differences exist, so that the detection of early novel coronaviruses by the antibody detection still has the possibility of false negative. Also, false positives may occur due to cross-reactivity.
Nucleic acids are the genetic material of viruses, and detection of sequences characteristic of viral sequences can be used to determine whether a viral infection has occurred. The nucleic acid detection has the characteristics of early diagnosis, high sensitivity and specificity and the like, and is the 'gold standard' for determining new coronary pneumonia. The most widely used nucleic acid detection means at present is the real-time fluorescent quantitative RT-PCR (q-PCR) technology, but the q-PCR detection has the following problems: one, false negatives may occur: because q-PCR detection is a point outline (only aiming at 2-3 sites on a virus genome and covering < 0.5% of the virus genome), whether a patient is positively infected or not is judged by judging whether a virus characteristic sequence with the length of more than one hundred base pairs corresponding to an amplification primer exists or not, detection failure is easily caused when the reasons such as target region variation, target region degradation, background interference and the like are met, improper sampling, improper specimen storage, adoption of different types of specimens and use of different manufacturer reagents can cause the occurrence of false negative in a nucleic acid detection result, so that missed diagnosis is caused; (II) low detection precision: the detection precision is 50-70% generally, so that the defects need to be compensated by two or more times of repeated detection; (III) the requirement on detection equipment or platforms is high: on one hand, the high-sensitivity q-PCR instrument is expensive; on the other hand, the requirements on cleanliness and operators in laboratories are also high. In addition, SARS-CoV-2, as an RNA virus, has a very high mutation rate, which may contribute to its spread and toxicity, and, due to rapid mutation, has many negative effects on the accuracy of its nucleic acid detection.
According to the information on the official network of the national drug administration, 23 novel coronavirus detection products are approved by the national drug administration in an emergency by 03-27.2020, wherein 15 novel coronavirus nucleic acid detection reagents and 8 antibody detection reagents are adopted. These novel coronavirus detection products do not overcome the above disadvantages of antibody detection or nucleic acid detection.
Furthermore, a research report on preprint website arXiv published by Tomoko Matsuda et al at 20.2.2020 (phenolic analysis of the section access priority syndrome 2 retrieved the section routes of introductions to Taiwan, the United States, and Japan) shows: the source of the novel coronavirus is controversial all the time, and if a positive sample is subjected to large-scale sequencing, the accumulated big data can really determine the source and the evolution direction of the virus; the source, the propagation path and the evolution direction of the virus are found out, and the method has great significance for the next prevention and control. This indicates that: the multi-sample and large-scale precise sequencing of SARS-CoV-2 virus will help to clear the source and evolution direction of the virus so as to guide the subsequent prevention and control work.
A study report on preprinted website medRxiv published by Lianlunjuan team, the Chinese institute of engineering, 4.14.2020 (patent-derived pathologies impact SARS-CoV-2, Handping Yao et al) states that: variation and diversity of the novel coronavirus strains are either greatly underestimated; the difference of different variant strains in cytopathic effect and viral load can reach 270 times; a trinucleotide mutation greatly enhances the replication rate and pathogenic capacity of the virus, and patients with this mutation were found to remain 45 days nucleic acid positive. This indicates that: SARS-CoV-2 virus detection requires not only determination of the presence or absence of SARS-CoV-2, but also accurate determination of the genetic variation of SARS-CoV-2, so as to guide subsequent isolation and treatment measures.
Next-Generation Sequencing (second Generation Sequencing) is an alternative method for detecting viral nucleic acid, and can obtain all information of viral genome sequence by Sequencing and analyzing the whole sequence base of the viral genome. However, the short read length of the second-generation sequencing results in great difficulty in assembling the whole genome sequence of the virus, and in order to improve the coverage and accuracy, a higher sequencing depth is required, thereby aggravating the disadvantages of high cost and long time of the second-generation sequencing. For this reason, the second generation sequencing technology is only suitable for scientific research of a small amount of samples, and is not suitable for large-scale routine detection. Indeed, there is currently no choice to apply second generation sequencing technology to screening SARS-CoV-2 virus for a broad population of people.
Accordingly, the technical problem to be solved by the present invention is to provide a novel method capable of detecting the presence of the novel coronavirus SARS-CoV-2 and, if present, determining the genomic sequence of SARS-CoV-2, which method is capable of achieving early diagnosis, convenience and rapidity, high throughput, high sensitivity, high specificity and high accuracy while reducing the false negative rate and the false positive rate.
Disclosure of Invention
The invention provides a gene chip which can be used for detecting whether a novel coronavirus SARS-CoV-2 exists or not and determining the genome sequence of SARS-CoV-2 if the novel coronavirus SARS-CoV-2 exists, the gene chip comprises 29846 groups of probes, each group of probes comprises 8 probes with the length of 25bp, the 8 probes cover the same section of the SARS-CoV-2 genome, the first probe in each group of probes is completely identical with the section of the SARS-CoV-2 genome, the second probe, the third probe and the fourth probe are different from the first probe at a specific site and are respectively three other bases in A, T, C, G at the specific site, the fifth probe, the sixth probe, the seventh probe and the eighth probe are respectively and completely complementary with the first probe, the second probe, the third probe and the fourth probe, the specific sites in each set of probes correspond to different sites in the SARS-CoV-2 genome and all the specific sites completely cover the 13 th-29858 th of the SARS-CoV-2 genome, and the specific site is the 13 th site in the probes.
The invention also provides a kit, which comprises the gene chip.
The present invention also provides a method which can be used to detect the presence of the novel coronavirus SARS-CoV-2 and, if present, to determine the genomic sequence of SARS-CoV-2, said method comprising the steps of:
(1) carrying out RNA extraction, reverse transcription, amplification, fluorescence labeling and fragmentation on an experimental sample to be detected;
(2) hybridizing the product of step (1) with the gene chip according to the present invention,
(3) determining whether the novel coronavirus SARS-CoV-2 exists in the experimental sample to be detected based on the hybridization signal of the probe on the gene chip and the product of the step (1) and determining the genome sequence of SARS-CoV-2 if the novel coronavirus SARS-CoV-2 exists.
The invention also relates to the use of the gene chip according to the invention or the kit according to the invention or the method according to the invention for the early warning of new viruses in the subfamily coronaviruses.
Drawings
FIG. 1 shows a schematic diagram of the probe design method of the present invention, in which specific sites (shown in bold and underlined) in each probe subgroup correspond to SARS-CoV-2 genome positions 146, 147, 148, 149, respectively. For example, to detect the 146 th base of SARS-CoV-2 genome, 8 probes of 25bp in length were designed, of which only 4 are shown in FIG. 1, and the other 4 are reverse-complementary to the 4 shown in FIG. 1; the specific site is located at the center of the probe, namely the 13 th site, the base of the first probe at the specific site is A (the same as the base at the 146 th site of SARS-CoV-2 genome), and the bases of the second probe, the third probe and the fourth probe at the specific site are different from each other and are C, T, G; the sequences of the first probe, the second probe, the third probe and the fourth probe are completely identical to the bases of the 134 th-145 and 147 th-158 th sites of SARS-CoV-2 genome except for the 146 th site corresponding to the specific site.
Fig. 2 shows: in one embodiment of the present invention, the result of the chip scan is obtained by fixing 238768 probes on the gene chip, and the experimental sample is a positive sample.
Fig. 3 shows: in one embodiment of the present invention, the result of the chip scan, wherein 238768 probes are immobilized on the gene chip, is the negative control of the experimental sample.
Detailed Description
Unlike the practical practice of using q-PCR technology universally and only a small amount of research work using second-generation sequencing technology to perform nucleic acid detection of the novel coronavirus SARS-CoV-2, the present inventors have creatively conceived of using a re-sequencing method to detect the presence or absence of the novel coronavirus SARS-CoV-2 and, if present, to determine the genomic sequence of SARS-CoV-2.
The re-sequencing chip is a special type gene chip, the detection result is judged through one or more continuous sequences, the final judgment is not influenced by local interference, the characteristic is very important under the conditions of low sample concentration, high background signal and complex detection sample environment, and the detection sensitivity can be greatly improved. In addition, the coverage rate of the re-sequencing chip is far greater than that of q-PCR (polymerase chain reaction) (< 0.5%), the coverage rate is improved by about 200 times, so that the detection sensitivity is greatly improved by improving the richness of acquired information, and when the CT value of q-PCR detection is greater than a threshold value to cause false negative of a detection result, whether a novel coronavirus signal exists or not can be distinguished by depending on the specificity of a chip probe, so that more accurate diagnosis can be made.
The invention utilizes the re-sequencing chip to carry out the accurate sequencing of the complete coverage of the SARS-CoV-2 virus genome, overcomes the defects of the known novel coronavirus SARS-CoV-2 detection method in the prior art, realizes the early diagnosis, is convenient and quick, has high flux, high sensitivity, high specificity and high accuracy, and simultaneously reduces the false negative rate and the false positive rate.
As used herein, the term "gene chip" refers to a chip obtained by using a large number of specific oligonucleotide fragments or gene fragments as probes and regularly immobilizing the oligonucleotide fragments or gene fragments on a solid support such as a silicon wafer, a glass plate, a plastic plate or a nylon substrate to form a two-dimensional array. The sequencing principle of the gene chip is a hybridization sequencing method, that is, a method of sequencing an experimental sample by hybridizing with a nucleic acid probe of a known sequence immobilized on the gene chip.
As used herein, the term "in situ synthesized gene chip" refers to a chip obtained by sequentially binding A, G, C, T four bases to the sequence of a substrate by an in situ photolithography synthesis method or an in situ jet printing synthesis method to synthesize a large number of desired oligonucleotide fragments or gene fragments as probes. The in-situ synthesized oligonucleotide chip has the advantages of high density, capability of synthesizing oligonucleotides with any sequence and the like, and is suitable for DNA sequence determination, SNP analysis and the like.
The orthocoronaviridae (scientific name: orthocoronaviridae) is known as Coronavirus (Coronavir), which belongs to the family of coronaviridae, the suborder coronaviridae, the order Neuroviridae, and is a zoonotic single-stranded RNA virus that is transmitted between animals and humans. The different virus species within the orthocoronaviridae subfamily share certain commonalities, e.g., genome sizes between 26,000 and 32,000 base pairs, which is the largest class of RNA viruses on the genomic scale; the capsule collagen fiber is spherical or elliptical under an electron microscope, and is provided with regularly arranged capsule collagen fiber protrusions which are shaped like a crown, so the capsule collagen fiber is named; the virus envelope is composed of double-layer lipid, membrane protein and spike protein are inserted, and some can also have hemagglutinin; the virus is internally provided with a nucleoprotein core consisting of RNA and capsid protein and has a spiral structure.
There are currently seven known classes of human coronaviruses, three of severe acute respiratory syndrome coronavirus (SARS-CoV), middle east respiratory syndrome coronavirus (MERS-CoV), and severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) can cause fatal respiratory diseases; the remaining four coronaviruses are common pathogens of the human common cold, and do not usually cause serious illness, but are limited to the possible complications of pneumonia in a few immunocompromised patients.
SARS-CoV-2 is an enveloped single-stranded, positive-stranded RNA virus with a genome length of 29903 bp. The 5' end of the genome carries a cap structure, which then contains 6-10 Open Reading Frames (ORFs). The first ORF occupying genome 2/3 encodes a replicase, and the other 1/3 of the genome encodes mainly structural proteins, typically including spike, S, minor envelope, E, envelope, M, and nucleoprotein (N). The E protein and the M protein are mainly involved in the assembly process of the virus, and the N protein wraps the genome to form a nucleoprotein complex.
The gene chip of the present invention is designed based on the re-sequencing of SARS-CoV-2 virus genome. The method utilizes the advantages of high probe density and large quantity of the chip to design a group of probes for each site of the viral genome to be sequenced (four probes with corresponding positions of A, T, G, C are designed for each base, four probes are respectively arranged on the positive chain and the negative chain, and therefore eight probes are totally arranged) to detect each base. When the target nucleic acid sequence with fluorescent label is hybridized with the probe on the gene chip, the corresponding positions respectively comprise A, T, G, C four probes or complete match hybridization or non-specific hybridization, thereby generating obvious brightness difference, and a group of probe sequences with completely complementary sequences are obtained by determining the probe position with the strongest fluorescence intensity, so that the sequence of the target nucleic acid can be determined.
Accordingly, the present invention provides a gene chip capable of detecting the presence or absence of the novel coronavirus SARS-CoV-2 and, if present, determining the genomic sequence of SARS-CoV-2, said gene chip comprising 29846 sets of probes, each set of probes comprising 8 probes of 25bp in length, the 8 probes covering the same segment of the SARS-CoV-2 genome, the first probe in each set of probes being identical to the segment of the SARS-CoV-2 genome, the second, third and fourth probes being different from the first probe only at one specific site and being the remaining three bases in A, T, C, G at the specific site, the fifth, sixth, seventh and eighth probes being different from the first, second, third and eighth probes, respectively, The fourth probe is completely complementary, the specific site in each set of probes corresponds to a different site in the SARS-CoV-2 genome and all the specific sites completely cover the 13 th-29858 th site of the SARS-CoV-2 genome, the specific site is the 13 th site in the probes.
The gene chip of the invention can at least realize 2 functions: (1) detecting the presence or absence of SARS-CoV-2;
(2) when the presence of SARS-CoV-2 is detected, the genomic sequence of SARS-CoV-2 can also be determined simultaneously.
Although the genome length of SARS-CoV-2 is 29903bp, the last 33 bases of the genome constitute the polyA tail, and therefore no probe is designed for these 33 sites; in addition, since the 13 th site in the probe is a site at which the first 12 bases and the last 12 bases of the genome sequence cannot be probed, the gene chip of the present invention comprises 29846 sets of probes (29903-33-12-12). Since each set of probes comprises 8 probes each having a length of 25bp, the gene chip of the present invention comprises 29846 (sets) × 8 (sets) ═ 238768 probes having a length of 25 bp.
The 8 probes comprised by each set of probes can be divided into 2 subgroups, with 4 probes in the first subgroup (consisting of the first, second, third and fourth probes) being fully complementary to 4 probes in the second subgroup (consisting of the fifth, sixth, seventh and eighth probes). The only difference between the four probes in the first/second subset is: one of A, T, G, C which differ from each other at a specific site.
As used herein, "specific site" refers to a first subset or a second subset of 4 probes in the same set that are different from each other at that site by one of A, T, G, C, but identical at other sites; the characteristic site is located at the center of the probe, i.e., position 13.
For example, a set of probes in the standard probes comprises 8 probes, each of the 8 probes covering positions X to (X +24) of the SARS-CoV-2 genome, the sequence of the first probe is identical to the sequence of positions X to (X +24) of the SARS-CoV-2 genome, if the first probe is A at a specific site (i.e., a site corresponding to position X +12 of the SARS-CoV-2 genome), the second probe, the third probe, and the fourth probe are one of T, G, C different from each other at the specific site, but identical to the first probe at the other 24 sites; the fifth probe, the sixth probe, the seventh probe and the eighth probe are completely complementary with the first probe, the second probe, the third probe and the fourth probe respectively. It should be noted that the above examples are only for better illustration of the present invention and do not represent any limitation to the present invention.
In the gene chip of the present invention, the specific sites in each group of probes correspond to different sites in SARS-CoV-2 genome, and all the specific sites cover the 13 th-29858 th site of SARS-CoV-2 genome completely. That is, 29846 specific sites among 29846 sets of probes on the biochip of the present invention correspond to 13 th to 29858 th sites of SARS-CoV-2 genome differently from each other, thereby enabling each site of SARS-CoV-2 genome to be sequenced accurately to achieve highly sensitive, highly accurate, and full-scale genome sequencing.
In some embodiments of the present invention, using the gene chip of the present invention, it is possible to complete the reading of the full-length SARS-CoV-2 virus genome sequence and obtain information about all known gene variations rapidly in one step. On the basis, the source and the propagation path of the virus can be analyzed and obtained by referring to a known virus variation evolutionary tree. Furthermore, by observing and summarizing the prognosis of patients with COVID-19 that corresponds to a particular viral gene variation, the trend of the evolution of toxicity of different variant viruses can be analyzed.
In one embodiment, the present invention is used to perform large-scale detection of patients who have been generally examined (e.g., by q-PCR nucleic acid detection or antibody detection) to show SARS-CoV-2 positivity, to obtain a large amount of the full-length genomic sequence and genetic variation information of SARS-CoV-2 virus. Based on the obtained deep genetic information and the big data of variation information, the virus toxicity evolution trend research, the source research, the propagation research and the like of the SARS-CoV-2 virus can be carried out, thereby providing valuable guide information for subsequent prevention, diagnosis and treatment.
In addition, because the gene chip of the invention adopts the re-sequencing technology, the detection is not influenced as long as the emerging new variant virus and the unknown virus belong to the coronavirus subfamily; in this case, an abnormal signal will appear at the normal sequencing position, so that a new virus warning can be obtained at the first time. Therefore, the gene chip is particularly suitable for continuously tracking and detecting the coronavirus subfamily of key epidemic propagation points with dense people streams such as vegetable farms, supermarkets, markets, stations, buses, subways, aircrafts and the like so as to realize the early discovery of the occurrence trend of malignant infectious diseases, thereby effectively guiding the rapid and accurate follow-up decision making.
In the embodiment of the present invention, the gene chip of the present invention may be any type of gene chip known in the art, such as an in situ synthesis gene chip, a spotting synthesis gene chip, a microbead gene chip, and the like. In some preferred embodiments of the present invention, the gene chip of the present invention is an in situ synthesis gene chip. Because the in situ synthesis gene chip can realize higher density and lower cost, and the more the samples are, the larger the sequencing scale is, the lower the average cost is, the great advantage is provided compared with the second generation sequencing technology which is only suitable for sequencing a small amount of samples and has high cost.
The invention also provides a kit, which comprises the gene chip.
In an embodiment of the invention, the kit comprises, in addition to the gene chip, reagents for performing RNA extraction and/or reverse transcription and/or amplification and/or fluorescent labeling and/or fragmentation and/or hybridization and/or staining and/or washing/rinsing.
As used herein, "reverse transcription" refers to a process of synthesizing DNA using RNA as a template. Since SARS-CoV-2 is an RNA virus, reverse transcription allows the synthesis of cDNA using the RNA genome of SARS-CoV-2 as a template.
As used herein, "fragmenting" refers to the process of cleaving/breaking DNA by enzymatic or ultrasonic means to bring the product within a certain length. In a preferred embodiment of the invention, the length of the fragmentation product is from 30 to 70bp, more preferably from 40 to 60bp, most preferably from 45 to 55 bp.
As used herein, "experimental sample" includes "biological samples" and "non-biological samples". "biological sample" refers to a sample such as sputum, nasopharyngeal swab, oropharyngeal swab, or alveolar lavage obtained from an organism (e.g., a human); when the organism is infected with SARS-CoV-2, the RNA of SARS-CoV-2 can be extracted from such a sample. "non-biological sample" refers to a sample taken from an environment (e.g., water, mop, doorknob, tabletop, floor, etc.); if SARS-CoV-2 is contained in the environment, SARS-CoV-2 RNA can be extracted from such a sample.
In addition, the present invention provides a method which can be used for detecting the presence of the novel coronavirus SARS-CoV-2 and, if present, determining the genomic sequence of SARS-CoV-2, said method comprising the steps of:
(1) carrying out RNA extraction, reverse transcription, amplification, fluorescence labeling and fragmentation on an experimental sample to be detected;
(2) hybridizing the product of step (1) with the gene chip according to the present invention,
(3) determining whether the novel coronavirus SARS-CoV-2 exists in the experimental sample to be detected based on the hybridization signal of the probe on the gene chip and the product of the step (1) and determining the genome sequence of SARS-CoV-2 if the novel coronavirus SARS-CoV-2 exists.
In embodiments of the invention, reverse transcription and amplification of the test sample to be tested may be performed separately or simultaneously. In some embodiments of the invention, reverse transcription and amplification are performed by a one-step process, i.e., reverse transcription and amplification are performed simultaneously in one step. In some embodiments of the invention, reverse transcription and amplification are performed separately by a two-step process, i.e., reverse transcription followed by amplification.
In a preferred embodiment of the invention, the length of the product of step (1) is 30-70bp, more preferably 40-60bp, most preferably 45-55 bp.
In addition, the invention also relates to the application of the gene chip or the kit or the method in the invention in the early warning of new viruses in the subfamily coronavirus.
Further, the present invention also includes embodiments described in the following aspects.
In a first aspect: a gene chip capable of being used for detecting the presence or absence of a novel coronavirus SARS-CoV-2 and, if present, determining the genomic sequence of SARS-CoV-2, said gene chip comprising 29846 sets of probes, each set of probes comprising 8 probes of 25bp in length, the 8 probes covering the same segment of the SARS-CoV-2 genome, the first probe in each set of probes being identical to the segment of the SARS-CoV-2 genome, the second, third and fourth probes being different from the first probe only at one specific site and being the remaining three bases in A, T, C, G at the specific site, the fifth, sixth, seventh and eighth probes being fully complementary to the first, second, third and fourth probes, respectively, the specific sites in each set of probes correspond to different sites in the SARS-CoV-2 genome and all the specific sites completely cover the 13 th-29858 th of the SARS-CoV-2 genome, and the specific site is the 13 th site in the probes.
In a second aspect: the gene chip according to the first aspect, wherein the gene chip is an in situ synthesis gene chip.
In a third aspect: a kit comprising a gene chip according to the first or second aspect.
In a fourth aspect: a method which can be used to detect the presence of the novel coronavirus SARS-CoV-2 and, if present, to determine the genomic sequence of SARS-CoV-2, said method comprising the steps of:
(1) carrying out RNA extraction, reverse transcription, amplification, fluorescence labeling and fragmentation on an experimental sample to be detected;
(2) hybridizing the product of step (1) with the gene chip according to the first aspect or the second aspect,
(3) determining whether the novel coronavirus SARS-CoV-2 exists in the experimental sample to be detected based on the hybridization signal of the probe on the gene chip and the product of the step (1) and determining the genome sequence of SARS-CoV-2 if the novel coronavirus SARS-CoV-2 exists.
In a fifth aspect: the method of the fourth aspect, wherein the length of the product of step (1) is 30-70bp, preferably 40-60bp, more preferably 45-55 bp.
A sixth aspect: use of a gene chip according to the first or second aspect or a kit according to the third aspect or a method according to the fourth or fifth aspect for the forewarning of new viruses within the subfamily coronaviridae.
The various embodiments and preferences described above for the individual components, steps and parts of the gene chip, kit, method, use according to the invention can be combined with one another (as long as they are not inherently contradictory to one another), and the various embodiments formed by this combination are considered part of the disclosure of the present application.
The technical solution of the present invention will be more clearly and clearly illustrated by way of example in the accompanying drawings and examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention in any way. The scope of the invention is only limited by the claims.
Examples
Unless otherwise indicated, the enzymes used in the following examples and their buffers were purchased from New England Biolabs (NEB). The gene chip used in the examples was obtained from Seikagaku technologies (Hangzhou) Ltd, and 238768 standard probes as described herein were immobilized on the chip. The general procedures in molecular biology can be found, for example, in the molecular cloning guidelines. The genomic sequence information for SARS-CoV-2 can be found in the National Center for Biotechnology Information (NCBI) reference sequence NC-045512.2. The sequence information of the primers and probes used in the examples is shown in table 1 below.
Table 1: sequence information of related primers and probes
Example 1: SARS-CoV-2 detection by two-step method using gene chip of the invention
1.1 Experimental samples
SARS-CoV-2 virus RNA samples from the disease control center in Zhejiang province were used as positive samples, and RNA expressed by normal human cells was used (Whole blood samples, MasterPure, Lucigen Co., Ltd.)TMComplete DNA and RNA Purification Kit extraction) as negative control.
1.2 reverse transcription
In the following experiment, the experimental environment, experimental equipment, and the like were cleaned with an RNase remover (Vazyme).
(1) The following ingredients were added to a nuclease-free microcentrifuge tube:
reverse transcription system 1: 12 μ L
| Composition (I) | Origin of origin | Volume of |
| dATP、dTTP、dGTP、dCTP(10mM) | SANGON BIOTECH (SHANGHAI) Co.,Ltd. | 1μL |
| N6-1 primer (50. mu.M) | SHANGHAI GENERAY BIOTECH Co.,Ltd. | 1μL |
| N6-3 primer (100nM) | SHANGHAI GENERAY BIOTECH Co.,Ltd. | 1μL |
| Total RNA (10ng/ul) | Zhejiang province disease control center | 9μL |
(2) The mixture was heated to 65 ℃ for 5min, then incubated on ice for 1 min;
(3) the contents of the tube were collected by flash centrifugation, then the following ingredients were added:
reverse transcription System 2: 7 μ L
| Composition (I) | Origin of origin | Volume of |
| 5X First-Strand Buffer | ThermoFisher | 4μL |
| DTT(0.1M) | ThermoFisher | 1μL |
| RNaseOUTTMRecombinant RNase Inhibitor(40units/μl) | ThermoFisher | 1μL |
| SuperScriptTMIII RT(200units/μl) | ThermoFisher | 1μL |
(4) Gently pipetting and mixing, incubating at 25 ℃ for 5min, then at 50 ℃ for 60min, and then heating at 70 ℃ for 15min to inactivate the reaction;
(5) adding 1 μ L RNase H (5U/. mu.L, NEB), digesting at 37 deg.C for 20min, and heating at 94 deg.C for 2min to inactivate the reaction;
(6) the contents of the tube were collected by flash centrifugation, then the following ingredients were added:
polymerization system: 25 μ L
| Composition (I) | Origin of origin | Volume of |
| 10×NEB Buffer 2 | NEB | 2μL |
| N6-5 primer (100nM) | SHANGHAI GENERAY BIOTECH Co.,Ltd. | 2.5μL |
| ss cDNA | The product of step (5) | 20μL |
| Klenow(3’-5’，Exo-)(5units/μl) | NEB | 0.5μL |
(7) The reaction was inactivated by gentle pipetting and incubation at 37 ℃ for 60min followed by heating at 75 ℃ for 20 min.
1.3PCR amplification
(1) Uniformly mixing the unfrozen reagent in a vortex mode, and performing instantaneous centrifugation; the following ingredients were then added to a 0.2mL PCR tube to prepare an amplification mix;
and (3) PCR system: 20 μ L
| Composition (I) | Origin of origin | Volume of |
| Biotin-11-dUTP(1mM) | ThermoFisher | 0.2μL |
| N6-2 primer (10. mu.M) | SHANGHAI GENERAY BIOTECH Co.,Ltd. | 1μL |
| ds DNA | Obtained as described in example 1.2 | 2μL |
| 2×PrimeSTAR Max | Takara | 10μL |
| dd H2O | SWAN | 6.8μL |
(2) Vortexing the prepared amplification mixture for several seconds, mixing, centrifuging instantaneously, and placing into a PCR instrument (BIO-RAD, T100)TMThermal Cycler) and set up the program as follows:
(3) detecting the PCR product by using 1% agarose gel;
(4) recovery was performed using a clean recovery Kit (FastPure Gel DNA Extraction Mini Kit, Vazyme) according to the manufacturer's instructions, and then the concentration of the recovered product was checked using Qubit4(Fluorometer, Invitrogen).
1.4 fragmentation of PCR products
(1) The recovered PCR products were mixed with the ingredients shown in the following table to prepare a fragmentation product mixture:
reaction system: 50 μ L
| Composition (I) | Origin of origin | Volume of |
| PCR product (40ng/ul) | Obtained as described in example 1.3 | 18μL |
| 10×DNase Reaction Buffer | ThermoFisher | 5μL |
| DNase I(1/100U/μL) | ThermoFisher | 2μL |
| dd H2O | SWAN | 25μL |
Note: preheating DNase I at 37 ℃ for 10min before adding the DNase I;
(2) vortex the prepared fragmentation mixture for several seconds, mix well, centrifuge instantaneously, and place into PCR instrument (BIO-RAD, T100)TMThermal Cycler) and set up the program as follows:
| temperature of | Time of day |
| 37℃ | 20min |
| 95℃ | 15min |
| 4℃ | Holding |
(3) The fragmentation product was detected on a 2% agarose gel and the results showed: the size of the fragmentation product is 50-100 bp.
(4) The concentration of the fragmented products was determined using Qubit4(Fluorometer, Invitrogen) and the results showed: the concentration of the fragmentation product was 20 ng/ul.
1.5 fragmentation product hybridization
1.5.1 prehybridization
1) Starting a hybridization box at 45 ℃ and preheating in advance for 30min (or until the temperature is stable);
3) adding 50ml of 6M NaCl saturated solution into a sealed hybridization box, and putting the sealed hybridization box and a hybridization plate into a 45 ℃ hybridization box to preheat for 30min in advance;
4) wash A and Wash B were prepared and the formulations were as follows.
| Wash A | 1L | Wash B | 1L |
| 20×SSC | 100mL | 20×SSC | 25mL |
| 10％Tween-20 | 1mL | 10％Tween-20 | 1mL |
| ddH2O | 899mL | ddH2O | 874mL |
1.5.2 hybridization
1) Hybridization mixtures were prepared as shown in the following table;
and (3) hybridization system: 65.35 μ L
| Composition (I) | Origin of origin | Volume of |
| 20X SSC | Invitrogen | 14μL |
| 10％Tween-20 | Sigma | 0.35μL |
| Cy3-AM1(10nM) | SHANGHAI GENERAY BIOTECH Co.,Ltd. | 1μL |
| Fragmented PCR product (20ng/ul) | Obtained as described in example 1.4 | 48μL |
2) Vortexing the hybridization mixed solution for 5s, mixing uniformly, performing instantaneous centrifugation, performing denaturation at 95 ℃ for 15min, cooling to 45 ℃, transferring the hybridization mixed solution to a position corresponding to the hybridization plate and the chip, buckling the chip plate, and checking to ensure that no bubbles are generated;
3) after confirming that no bubble is present, the cells were put into a hybridization chamber at 45 ℃ for hybridization for 2 hours.
Note: the hybridization plate was incubated at 45 ℃ on a PCR instrument while the sample was being applied.
1.5.3 cleaning
1) Preparation work:
a) opening a 39 ℃ hybridization box to preheat for 30min in advance (or until the temperature is stable);
b) 20mL of Wash A and a cleaning plate are placed in a 25 ℃ hybridization box (room temperature) to be preheated for 10 min;
c) 20mL of Wash B and Wash plate were pre-heated in a 39 ℃ hybridization chamber for 10 min.
2) And (3) cleaning operation:
a) after hybridization, taking the chip plate and the hybridization plate out of the sealed hybridization box, and transferring the chip plate to a 20mL Wash A plate for incubation for 3 min; the speed is very fast when the chip is transferred, and the surface of the chip is prevented from drying;
b) after 3min, the chip plate was removed, placed in a Wash plate containing 20mL of Wash B, and incubated at 39 ℃ for 3 min.
1.5.4 dyeing
1) 50 μ L of Stain 1(SAPE Stain (ex Invitrogen)) was added to the staining plate 1 at the corresponding position on the chip;
2) taking the chip plate out of the Wash B, placing the chip plate in a staining plate 1, and incubating for 10min at room temperature (25 ℃);
3) the chip plate was removed from the stained plate 1 and placed in a Wash plate containing 20mL of Wash A and incubated for 5min at room temperature (25 ℃).
1.5.5 chip Scan imaging
After rinsing once with 1mL of 4 XSSC, fluorescence signal detection was performed using a SUMMIT chip scanner (available from Promega technologies, Hangzhou) Inc.) and the scan was read using the VirusHunter analysis software.
The chip scanning result of the positive sample is shown in FIG. 2, and the measured specific sequence information is shown in SEQ ID No. 6. The alignment of SEQ ID No.6 with the NCBI reference sequence NC-045512.2 (bases 13-29858) shows: the coverage is 99.9%, and the consistency is 99.3%, thereby proving that the gene chip of the invention has high flux, high sensitivity and high accuracy. The results of the chip scan for the negative control are shown in FIG. 3. As is apparent from FIG. 3, the fluorescence intensity on the chip is very weak because normal human RNA not infected with SARS-CoV-2, after undergoing reverse transcription, amplification and fragmentation, cannot non-specifically hybridize with the gene chip of the present invention, and thus its presence does not interfere with the detection of SARS-CoV-2, thereby demonstrating that the gene chip of the present invention has high specificity.
While particular embodiments of the present invention have been illustrated and described, it is not intended that these embodiments illustrate and describe all possible forms of the invention. Rather, the words used in the specification are words of description rather than limitation. It will be apparent to those skilled in the art that various other changes and modifications can be made without departing from the general scope of the disclosure. It is therefore intended to cover in the appended claims all such changes and modifications that are within the scope of this invention.
Sequence listing
<110> Shengjie science and technology (Hangzhou) Co., Ltd
Shengjie science and technology (Jiaxing) Co., Ltd
<120> a novel coronavirus detection gene chip
<130> D-CF200241
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 26
<212> DNA
<213> Artificial sequence ()
<220>
<221> misc_feature
<222> (21)..(26)
<223> n is a, c, g, or t
<400> 1
gccggagctc tgcagatatc nnnnnn 26
<210> 2
<211> 20
<212> DNA
<213> Artificial sequence ()
<400> 2
gccggagctc tgcagatatc 20
<210> 3
<211> 40
<212> DNA
<213> Artificial sequence ()
<400> 3
gccggagctc tgcagatatc tttttttttt tttttttttt 40
<210> 4
<211> 30
<212> DNA
<213> Artificial sequence ()
<400> 4
gccggagctc tgcagatatc attaaaggtt 30
<210> 5
<211> 16
<212> DNA
<213> Artificial sequence ()
<400> 5
tcgtagctag cgtact 16
<210> 6
<211> 29846
<212> DNA
<213> sequencing result of Positive sample ()
<220>
<221> misc_feature
<222> (3)..(4)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (7)..(7)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (37)..(37)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (48)..(48)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (124)..(124)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (126)..(128)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (223)..(223)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (2471)..(2471)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (2473)..(2474)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (2479)..(2479)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (3277)..(3277)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (3479)..(3480)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (4025)..(4025)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (4528)..(4528)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (4612)..(4612)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (4859)..(4860)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (4864)..(4864)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (4892)..(4892)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (4901)..(4902)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (4940)..(4940)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (5014)..(5015)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (5017)..(5017)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (5103)..(5104)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (5107)..(5107)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (5110)..(5110)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (5272)..(5272)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (5737)..(5737)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (5739)..(5739)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (5741)..(5743)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (5858)..(5858)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (6086)..(6086)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (6090)..(6090)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (6236)..(6236)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (6706)..(6706)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (6733)..(6733)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (7602)..(7602)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (7812)..(7812)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (8513)..(8513)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (8515)..(8515)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (8584)..(8584)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (8936)..(8936)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (9350)..(9350)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (9731)..(9731)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (12937)..(12937)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (12945)..(12945)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (13130)..(13130)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (13152)..(13153)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (13270)..(13272)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (14840)..(14840)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (14946)..(14947)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (15360)..(15360)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (15626)..(15626)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (16082)..(16082)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (17851)..(17852)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (17855)..(17855)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (18369)..(18369)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (19119)..(19121)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (19125)..(19125)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (20295)..(20295)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (20531)..(20531)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (20535)..(20535)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (21402)..(21402)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (21404)..(21404)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (22383)..(22383)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (22528)..(22529)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (22633)..(22633)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (22900)..(22900)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (23343)..(23343)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (23768)..(23769)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (24238)..(24239)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (24241)..(24241)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (24248)..(24249)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (24251)..(24251)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (24377)..(24377)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (25856)..(25856)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (25987)..(25987)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (26036)..(26036)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (26082)..(26083)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (26209)..(26209)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (26473)..(26473)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (26476)..(26476)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (26549)..(26549)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (26579)..(26580)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (26582)..(26584)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (27330)..(27331)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (27335)..(27335)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (27663)..(27663)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (27708)..(27708)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (27710)..(27710)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (29837)..(29837)
<223> n is a, c, g, or t
<400> 6
tgnntcntta atctcccccc caacgcgtgt agcctanaaa ctggcctnat tcgatactag 60
aacttcaaaa tcactgtggc cgtgattcgg ctgcatcctt agtggccaca cgcaatataa 120
ttgnannngc attaccgcgc aaggcaggac acgagcggct cgtttatctt ccgcaggatg 180
cttacgattt cgaccgcgtt gcagccgatc atgagcacat ctnggtttcg tcagtgtgcg 240
agcgaggggg gatacggaac gcattgtgcc tagttagtac gagaaaacaa acgtccaact 300
cagattcatc aatttacagc ttcgcggggt gctcgtacgt cggtttggag gagacgtgga 360
ggaggactta tcagaggcac gtcaacatct tcgagatggc acttgtggct tagtagaagt 420
tgaaaaaggc gttttgcctc aacttgaaca gccctatgtg ttcatcaaac gttcggatgc 480
tcgtactgca cctcaaggtc atgttatggt tgagctggta gcagaactcg aaggcattca 540
gtacggtcgt agtggtgaga cacttggtgt ccttgtccct catgtgggcg aaataccagt 600
ggcttaccgc aaggttcttc ttcgtaagaa cggtaataaa ggagctggtg gccatagtta 660
cggcgccgat ctaaagtcag ttgacttagg cgacgagctt ggcactgatc cttatgaaga 720
ttttcaagaa aactggaaca ctaaacatag cagtggtgtt acccgtgaac tcatgcgtga 780
gcttaacgga ggggcataca ctcgctatgt cgataacaac ttctgtggcc ctgatggcta 840
ccctcttgag tgcattaaag accttctagc acgtgctggt aaagcttcat gcactttgtc 900
cgaacaactg gactttattg acactaagag gggtgtatac tgctgccgtg aacatgagca 960
tgaaattgct tggtacacgg aacgttctga aaagagctat gaattgcaga caccttttga 1020
aattaaattg gcaaagaaat ttgacacctt caatggggaa tgtccaaatt ttgtatttcc 1080
cttaaattcc ataatcaaga ctattcaacc aagggttgaa aagaaaaagc ttgatggctt 1140
tatgggtaga attcgatctg tctatccagt tgcgtcacca aatgaatgca accaaatgtg 1200
cctttcaact ctcatgaagt gtgatcattg tggtgaaact tcatggcaga cgggcgattt 1260
tgttaaagcc acttgcgaat tttgtggcac tgagaatttg actaaagaag gtgccactac 1320
ttgtggttac ttaccccaaa atgctgttgt taaaatttat tgtccagcat gtcacaattc 1380
agaagtagga cctgagcata gtcttgccga ataccataat gaatctggct tgaaaaccat 1440
tcttcgtaag ggtggtcgca ctattgcctt tggaggctgt gtgttctctt atgttggttg 1500
ccataacaag tgtgcctatt gggttccacg tgctagcgct aacataggtt gtaaccatac 1560
aggtgttgtt ggagaaggtt ccgaaggtct taatgacaac cttcttgaaa tactccaaaa 1620
agagaaagtc aacatcaata ttgttggtga ctttaaactt aatgaagaga tcgccattat 1680
tttggcatct ttttctgctt ccacaagtgc ttttgtggaa actgtgaaag gtttggatta 1740
taaagcattc aaacaaattg ttgaatcctg tggtaatttt aaagttacaa aaggaaaagc 1800
taaaaaaggt gcctggaata ttggtgaaca gaaatcaata ctgagtcctc tttatgcatt 1860
tgcatcagag gctgctcgtg ttgtacgatc aattttctcc cgcactcttg aaactgctca 1920
aaattctgtg cgtgttttac agaaggccgc tataacaata ctagatggaa tttcacagta 1980
ttcactgaga ctcattgatg ctatgatgtt gacatctgat ttggctacta acaatctagt 2040
tgtaatggcc tacattacag gtggtgttgt tcagttgact tcgcagtggc taactaacat 2100
ctttggcact gtttatgaaa aactcaaacc cgtccttgat tggcttgaag agaagtttaa 2160
ggaaggtgta gagtttctta gagacggttg ggaaattgtt aaatttatct caacctgtgc 2220
ttgtgaaatt gtcggtggac aaattgacac ctgtgcaaag gaaattaagg agagtgttca 2280
gacattcttt aagcttgtaa ataaattttt ggctttgtgt gctgactcta tcattattgg 2340
tggagctaaa cttaaagcct tgaatttagg tgaaacattt gtcacgcact caaagggatt 2400
gtacagaaag tgtgttaaat ccagagaaga aactggccta ctcatgcctc taaaagcccc 2460
aaaagaaatt ntnnaaggng agggagaaac acttcccaca gaagtgttaa cagaggaagt 2520
tgtcttgaaa actggtgatt tacaaccatt agaacaacct actagtgaag ctgttgaagc 2580
tccattggtt ggtacaccag tttgtattaa cgggcttatg ttgctcgaaa tcaaagacac 2640
agaaaagtac tgtgcccttg cacctaatat gatggtaaca aacaatacct tcacactcaa 2700
aggcggtgca ccaacaaagg ttacttttgg tgatgacact gtgatagaag tgcaaggtta 2760
caagagtgtg aatatcactt ttgaacttga tgaaaggatt gataaagtac ttaatgagaa 2820
gtgctctgcc tatacagttg aactcggtac agaagtaaat gagttcgcct gtgttgtggc 2880
agatgctgtc ataaaaactt tgcaaccagt atctgaatta cttacaccac tgggcattga 2940
tttagatgtg tggagtatgg ctacatacta cttatttgat gagtctggtg agtttaaatt 3000
ggcttcacat atgtattgtt ctttctaccc cccagatgag gatgaagaag aaggtgattg 3060
tgaagaagaa gagtttgagc catcaactca atatgagtat ggtactgaag atgattacca 3120
atacaaacct ttggaatttg gtgccacttc tgctgctctt caacctgaag aagagcaaga 3180
agaagattgg ttagatgatg atagtcaaca aactgtcggt caacaagacg gcagtgagga 3240
caatcagaca actactattc aaacaattgt tgaggtncat cctcaattag agatggaact 3300
tacaccagtt gttcagacta ttgaagtgaa tagttttagt ggttatttaa aacttactga 3360
caatgcacaa gataaaaatg cagacattgt ggaagaagct aaaaaggtaa aaccaacagt 3420
ggttgttaat gcagccaatg tttaccttta acatggagga ggtgttgcag gagcctaann 3480
gatggctact aacaatgcca tgcaagttga atctgatgat tacatagcta ctaatggacc 3540
acgtaaagtg ggtggtagtt gtgttttaag cggacacaat cttgctaaac actgtcttca 3600
tgttgtcggc ccaaatgtta acaaaggtga agacattcaa cttcttaaga gtgcttatga 3660
aaattttaat cagcacgaag ttctacttgc accattatta tcagctggta tttttggtgc 3720
tgaccctata cattctttaa gagtttgtgt agatactgtt cgcacaaatg tctacttagc 3780
tgtctttgat aaaaatctct atgacaaact tgtttcaagc tttttggaaa tgaagagtga 3840
aaagcaagtt gaacaaaaga tcgctgagat tcctacggag gaagttaagc catttataac 3900
tgaaagaaaa ccttcagttg aacagagaaa acaagatgat aagaaaatca aagcttgtgt 3960
tgaagaagtt acaacaactc tggaagaaac taagttcctc acagaaaact tgttacttta 4020
tattnactac catggcaatc ttcatccaga ttctgccact cttgttagtg acattgacat 4080
cactttctgc aagaaagatg ctccatatat agtgggtgat gttgttcaag agggtgtttt 4140
aactgctgtg gttataccta ctaaaaaggc tggtggcact actgaaatgc tagcgaaagc 4200
tttgagaaaa gtgccaacag acaattatat aaccacttac gcgggtcagg gtttaaatgg 4260
ttacactggg gaggaggcaa agacagtgct taaaaagtgt aaaagtgcct tttacattct 4320
accatctatt atcttgaatg agaagcaaga aattcttgga actgtttctt ggaatttgcg 4380
agaaatgctt gcacatgcag aagaaacacg caaattaatg cctgtctgtg tggaaactaa 4440
agccatagtt tcaactatac agcgtaaata taagggtact aaaatacaag agggtgtggt 4500
tgattatggt gctagatttt acttttcngc aagcaaaaca actgtagcgt cacttatcaa 4560
cacacttaac gatctaaatg aaactcttgt tacaatgcca cttggctagg cnacacatgg 4620
cttaaatttg gaagacgctg ctcggtatat gagctctctc aaagtgccag ctacagtttc 4680
tgtttcttca cctgatgctg ttacagcgta taatggttat cttacttctt cttctaaaac 4740
acctgaagaa cattttattg aaaccatctc acttgctggt tcctataaag attggtccta 4800
ttctggacaa tctacacaac taggtataga atttcttaag agaggtgata aaagtgttnn 4860
aaanactagt aatcctacca cattccacct anatggtgca nngatcacct ttgacaatct 4920
taagacactt ctttctgtgn gagaagtgag gactattaag gtgtttacat cagtagacaa 4980
cattaacctc cacacgcaag ttgtggacat gganntnaca tatggacaac agtttggtcc 5040
aactgatttg gatggagctg atgttactaa aataaaacct cataattcac atgaaggtaa 5100
ttnnggntcn cttttaccta atgatgacac tctacgtgtt gaggcttttg agtactacca 5160
cacaactgat cctagttttc tgggtaggta catgtcagca ttaaatcaca cgaaaaagtg 5220
gaaataccca caagttaatg cgttcacttc tattaaatgg gcagataaca cnagttatct 5280
tgccactgca ttgttaacac tccaacaaat agagttgaag tttaatccac ctgctctaca 5340
agatgcttat tacagagcaa gggctggtga tgctgctaac ttttgtgcac ttatcttagc 5400
ctactgtaat aagacagtag gtgagttagg tgatgttaga gaaacaatga gttacttgtt 5460
ccaacatgcc aatttagatt cttgcaaaag agtcttgaac gtggtgtgta aaacttgtgg 5520
acaacagcag acaacccttt agggtgtaga agctgttatg tacatgggca cactttctta 5580
tgaacaattt aagaaaggtg ttcagatacc ttgtacgtgt ggtaaacaag ctacaaaata 5640
tctagtacaa caggagtcac cttttgttat gatgtcagca ccacctgctc agtatgaact 5700
taagcatggt acatttactt gtgctagtga gtacacngna nnnttgcagt gtggtcacta 5760
taaacatata acttctaaag aaactttgta ttgcatagac ggtgctttac ttccaaagtc 5820
ctcagaatac aaaggtccta ttacggatgt tttctacnga gaaaacagtt acacaacaac 5880
cataaaacca gttacttata aattggatgg tgttgtttgt acagaaattg accctaagtt 5940
ggacaattat tataagaaag ccaattctta tttcacagag caaccaattg atcttgtacc 6000
aaaccaacca tatccaaacg caagcttcga taattttaag tttgtatgtg atgatatcaa 6060
atttgctgat gatttaaacc atctgnaccn ttataagaaa cctgcttcaa gagagcttaa 6120
agttacattt ttccctgact taaatggtgg tgtggtggct attgattata aacactacac 6180
accctctttt aagaaaggag ctaaattgtt acataaacct attgtttggc atgacnataa 6240
tgcaactaat aaagccacgt ataaaccaaa tacctggtgt atacgttgtc tttggagcac 6300
aaaaccagtt gaaacatcaa tttcgtttga tgtactgagg tcggaggacg cgcagggaat 6360
ggataatctt gcctgcgaag atctaaaacc agtctctgaa gaagtagtgg aaaatcctac 6420
catacagaaa gacgttcttg agtgtaatgt gaaaactacc gaagttgtag gagacattat 6480
acttaaacca gcaaataata gtttaaaaat tacagaagag gttggccaca cagatctaat 6540
ggctgcttat gtagacaatt ctagtcttac tattaagaaa cctaatgaat tatctagagt 6600
attaggtttg aaaacccttg ctactcatgg tttagctgct gttaatagtg tcccttggga 6660
tactatagct aattatgcta agccttttct taacaagctt gtcgcnctaa caaccaacat 6720
agttacacgg tcngatgacc gtgtttgtac taattatatg ccttatttct ttactttatt 6780
gctacaattg tgtactttta ctagaagtac cacttctaga attaaagcat ctatgccgac 6840
tactatagca aagaatactg ttaagagtgt cggtaaattt tgtctagagg cttcatttaa 6900
ttatttgaag tcacctaatt tttctaaact gataaatatt ataatttggt ttttactatt 6960
aagtgtttgc ctaggttctt taatctactc aaccgctgct ttaggtgttt taatgtctaa 7020
tttaggcgtg ccttcttact gtactggtta cagagaaggc tatttgaact ctactaatgt 7080
cactattgca acctactgta ctggttctat accttgtagt gtttgtctta gtggtttaga 7140
ttctttagac acctatcctt ctttagaaac tatacaaatt accatttcat cttttaaatg 7200
ggatttaact gcttttggct tagttgcaga gtggtttttg gcatatattc ttttcactag 7260
gtttttctat gtacttggat tggctgcaat catgcaattg tttttcagct attttgcagt 7320
acattttatt agtaattctt ggcttatgtg gttaataatt aatcttgtac aaatggcccc 7380
gatttcagct atggttagaa tgtacatctt ctttgcatca ttttattatg tatggaaaag 7440
ttatgtgcat gttgtagacg gttgtaattc atcaacttgt atgatgtgtt acaaacgtaa 7500
tagagcaaca agagtcgaat gtacaactat tgttaatggt gttagaaggt ccttttatgt 7560
ctatgctaat ggaggtaaag gcttttgcaa actacacaat tncggttgtg ttaattgtga 7620
tacattctgt gctggtagta catttattag tgatgaagtt gcgagagact tgtcactaca 7680
gtttaaaaga ccaataaatc ctactgacca gtcttcttac atcgttgata gtgttacagt 7740
gaagaatggt tccatccatc attactttga taaagctggt caaaagactt atgaaagaca 7800
ttctctctct cnatttgtta acttagacaa cctgagagct aataacacta aaggttcatt 7860
gcctattaat gttatagttt ttgatggtaa atcaaaatgt gaagaatcat ctgcaaaatc 7920
agcgtctgtt tactacagtc agcttatgtg tcaacctata ctgttactag atcaggcatt 7980
agtgtctgat gttggtgata gtgcggaagt tgcagttaaa atgtttgatg cttacgttaa 8040
tacgttttca tcaactttta acgtaccaat ggaaaaactc aaaacactag tcgcaactgc 8100
agaagctgaa cttgcaaaga atgtgtcctt agacgatgtc ttatctactt ttatttcagc 8160
agctcggcaa gggtttgttg attcagatgt agaaactaaa gatgttgttg aatgtcttaa 8220
attgtcacat caatctgaca tagaagttac tggcgatagt tgtaataact atatgctcac 8280
ctataacaaa gttgaaaaca tgacaccccg tgaccttggt gcttgtattg actgtagtgc 8340
gcgtcatatt aatgcgcagg tagcaaaaag tcacaacatt gctttgatat ggaacgttaa 8400
agatttcatg tcattgtctg aacaactacg aaaacaaata cgtagtgctg ctaaaaagaa 8460
taacttacct tttaagttga catgtgcaac tactagacaa gttgttaatg tgncnacaac 8520
aaagatagca cttaagggtg gtaaaattgt taataattgg ttgaagcagt taattaaagt 8580
tacncttgtg ttcctttttg ttgctgctat tttctattta ataacacctg ttcatgtcat 8640
gtctaaacat actgactttt caagtgaaat cataggatac aaggctattg atggtggtgt 8700
cactcgtgac atagcatcta cagatacttg ttttgctaac aaacatgctg attttgacac 8760
atggtttagt cagcgtggtg gtagttatac taatgacaaa gcttgcccat tgattgctgc 8820
agtcataaca agagaagtgg gttttgtcgt gcctggtttg cctggcccga tattacgcac 8880
aactaatggt gactttttgc atttcttacc tagagttttt agtgcagttg gtaacnatag 8940
tcacacacca tcaaaactta tagagtacac tgactttgca acatcagctt gtgttttggc 9000
tgctgaatgt acaattttta aagatgcttc tggtaagcca gtaccatatt gttatgatac 9060
caatgtacta gaaggttctg ttgcttatga aagtttacgc cctgacacac gttatgtgct 9120
catggatggc tctattattc aatttcctaa cacctacctt gaaggttctg ttagagtggt 9180
aacaactttt gattctgagt acggtaggca cggcacttgt gaaagatcag aagctggtgt 9240
ttgtgtgtct actagtggta gatgggtact taacaatgat tattacagat ctttaccagg 9300
agttttctgt ggtgtagatg ctgtaaattt acttactaat atgtttacan caaaaatgca 9360
atttattggt gctttggaca tatcagcatc tatagtagct ggtggtattg tagctatcgt 9420
agtaacatgc cttgcctact attttatgag gtttagaaga gcttttggtg aatacagtca 9480
tgtagttgcc tttaatactt tactattcct tatgtcattc actgtactct gtttaacacc 9540
agtttactca ttcttacctg gtgtttattc tgttatttac ttgtacttga cattttatct 9600
tactaatgat gtttcttttt tagcacatat tcagtggatg gttatgttca cacctttagt 9660
acctttctgg ataacaattg cttatatcat ttgtatttcc acaaagcatt tctattggtt 9720
ctttagtaat ntcctaaaga gacgtgtagt ctttaatggt gtttccttta gtacttttga 9780
agaagctgcg ctgtgcacct ttttgttaaa taaagaaatg tatctaaagt tgcgtagtga 9840
tgtgctatta cctcttacgc aatataatag atacttagct ctttataata agtacaagta 9900
ttttagtgga gcaatggata caactagcta cagagaagct gcttgttgtc atctcgcaaa 9960
ggctctcaat gacttcagta actcaggttc tgatgttctt taccaaccac cacaaacctc 10020
tatcacctca gctgttttgc agagtggttt tagaaaaatg gcattcccat ctggtaaagt 10080
tgagggttgt atggtacaat taacttgtgg tacaactaca cttaacggtc tttggcttga 10140
tgacgtagtt tactgtccaa gacatgtgat ctgcacctct gaagacatgc ttaaccctaa 10200
ttatgaagat ttactcattc gtaagtctaa tcataatttc ttggtacagg ctggtaatgt 10260
tcaactcagg gttattggac attctatgca aaattgtgta cttcagctta aggttgatac 10320
agccaatcct aagacaccta agtataagtt tgttcgcatt caaccaggac agactttttc 10380
agtgttagct tgttacaatg gttcaccagt tggtgtttac caatgtgcta tgaggcccaa 10440
tttcactatt aagggttcat tccttaatgg ttcatgtggt agtgttggtt ttaacataga 10500
ttatgactgt gtctcttttt gttacatgca ccatatggaa ttaccaactg gagttcatgc 10560
tggcacagac ttagaaggta acttttatgg accttttgtt gacaggcaaa cagcacaagc 10620
agctggcacg gacacaacta ttacagttaa tgttttagct tggttgtacg ctgctgttat 10680
aaatggagac aggtggtttc tcaatcgatt taccacaact cttaatgact ttaaccttgt 10740
ggctatgaag tacaattatg aacctctaac acaagaccat gttgacatac taggacctct 10800
ttctgctcaa actggaattg ccgttttaga tatgtgtgct tcattaaaag aattactgca 10860
aaatggaatg aatggacgta ccatattggg tagtgcttta ttagaagatg aatttacacc 10920
ttttgatgtt gttagacaat gctcaggtgt tactttccaa agtgcagtga aaagaacaat 10980
caagggcaca caccactggt tgttactcac aattttgact tcacttttag ttttagtcca 11040
gagtactcaa tggtctttgt tctttttttt gtatgaaaat gcctttttac cttttgctat 11100
gggtattatt gctatgtctg cttttgcaat gatgtttgcg gaacataagc atgcatttct 11160
ctgtttgttt ttgttacctt ctcttgccac tgtagcttat tttaatatgg tctatatgcc 11220
tgctagttgg gtgatgcgta ttatgacatg gttggatatg gttgatacta gtttgtctgg 11280
ttttaagcta aaagactgtg ttatgtatgc atcagctgta gtgttactaa tccttatgac 11340
agcaagaact gtgtatgatg atggtgctgg gagagtgtgg acacttatga atgtcttgac 11400
actcgtttat aaagtttatt atggtaatgc tttagatcaa gccatttcca tgtgggctct 11460
tataatctct gttacttcta actactcagg tgtagttaca actgtcatgt ttttggccag 11520
aggtattgtt tttatgtgtg ttgagtattg ccctattttc ttcataactg gtaatacacg 11580
ctagtgtata atgctagttt attgtttctt aggctatttt tgtacttgtt actttggcct 11640
cttttgttta ctcaaccgct actttagact gactcttggt gtttatgatt acttagtttc 11700
tacacaggag tttagatata tgaattcaca gggactactc ccacccaaga atagcataga 11760
tgccttcaaa ctcaacatta aattgttggg tgttggtggc aaaccttgta tcaaagtagc 11820
cactgtacag tctaaaatgt cagatgtaaa gtgcacatca gtagtcttac tctcagtttt 11880
gcaacaactc agagtagaat catcatctaa attgtgggct caatgtgtcc agttacacaa 11940
tgacattctc ttagctaaag atactactga agcctttgaa aaaatggttt cactactttc 12000
tgttttgctt tccatgcagg gtgctgtaga cataaacaag ctttgtgaag aaatgctgga 12060
caacagggca accttacaag ctatagcctc agagtttagt tcccttccat catatgcagc 12120
ttttgctact gctcaagaag cttatgagca ggctgttgct aatggtgatt ctgaagttgt 12180
tcttaaaaag ttgaagaagt ctttgaatgt ggctaaatct gaatttgacc gtgatgcagc 12240
catgcaacgt aagttggaaa agatggctga tcaagctatg acccaaatgt ataaacaggc 12300
tagatctgag gacaagaggg caaaagttac tagtgctatg cagacaatgc ttttcactat 12360
gcttagaaag ttggataatg atgcactcaa caacattatc aacaatgcaa gagatggttg 12420
tgttcccttg aacataatac ctcttacaac agcagccaaa ctaatggttg tcataccaga 12480
ctataacaca tataaaaata cgtgtgatgg tacaacattt acttatgcat cagcattgtg 12540
ggaaatccaa caggttgtag atgcagatag taaaattgtt caacttagtg aaattagtat 12600
ggacaattca cctaatttag catggcctct tattgtaaca gctttaaggg ccaattctgc 12660
tgtcaaatta cagaataatg agcttagtcc tgttgcacta cgacagatgt cttgtgctgc 12720
cggtactaca caaactgctt gcactgatga caatgcgtta gcttactaca acacaacaaa 12780
gggaggtagg tttgtacttg cactgttatc cgatttacag gatttgaaat gggctagatt 12840
ccctaagagt gatggaactg gtactatcta tacagaactg gaaccacctt gtaggtttga 12900
tacagacaca cctaaaggtc ctaaagtgaa gtaatgngag cttantcaag gattaaacaa 12960
cctaaataga ggtatggtac ttggtagttt agctgccaca gtacgtctac aagctggtaa 13020
tgcaacagaa gtgcctgcca attcaactgt attatctttc tgtgcatttg ctgtagatgc 13080
tgctaaagct tacaaagatt atctagctag tgggggacaa ccaatcactn attgtgttaa 13140
gatgttgtac gnncacactg gtactggtca ggcaataaca gttacaccgg gagccaatat 13200
ggatcaagaa tcctttggtg gtgcatcgtg ttgtctgtgc tgccgttgcc acatagatca 13260
tccaaatccn nnaggatttt gtgacttaaa aggtaagtat gtacaaatac ctacaacttg 13320
tgctaatgac cctgtgggtt ttacacttaa aaacacagtc tgtaccgtct gcggtatgtg 13380
gaaaggttat ggctgtagtt gtgatcaact ccgcgaaccc atgcttcagt cagctgaagc 13440
acaatcgttt ttaaacgggt ttgcggtgta agtgcagccc gtcttacacc gtgcggcaca 13500
ggcactagta ctgatgtcgt atacagggct tttgacatct acaatgataa agtagctggt 13560
ttggctaaat tcctaaaaac taattgttgt cgcttccaag aaaaggacga agatgacaat 13620
ttaattgatt cttactttgt agttaagaga cacactttct ctaactacca acatgaagaa 13680
acaatttata atttacttaa ggattgtcca gctgttgcta aacatgactt ctttaagttt 13740
agaatagacg gtgacatggt accacatata tcacgtcaac gtcttactaa atacacaatg 13800
gcagacctcg gctatgcttt aaggcatttt gatgaaggta attgtgacac attaaaagaa 13860
atacttgtca catacacttg ttgtgatgat gattatttca ataaaaagga ctggtatgat 13920
tttgtagaaa acccagatat agtaacaaca tacgccaact taggtgaacg tgtacgccaa 13980
gctttgttaa aaacagtaca attctgtgat gccatgcgaa atgctggtat tgttggtgta 14040
ctgacattag ataatcaaga tctcaatggt aactggtatg atttcggtga tttcatacaa 14100
accacgccag gtagtggagt tcctgttgta gattcttatt attcattgtt aatgcctata 14160
ttaaccttga ccagggcttt aactgcagag tcacatgttg acactgactt aacaaagcct 14220
tacattaagt gggatttgtt aaaatatgac ttgacggaag agaggttaaa actctttgac 14280
cgttatttta aatattggga tcagacatac cacccaaatt gtgttaactg tttggatgac 14340
agatgcattc tgcattgtgc aaactttaat gttttattct ctacagtgtt cccacctaca 14400
agttttggac cactagtgag aaaaatattt gttgatggtg ttccatttgt agtttcaact 14460
ggataccact tcagagagct aggtgttgta cataatcagg atgtaaactt acatagctct 14520
agacttagtt ttaaggaatt acttgtgtat gctgctgacc ctgctatgca cgctgcttct 14580
ggtaatcttt cactagataa acgcactacg tgcttttcag tagctgcact tactaacaat 14640
gttgcttttc aaactgtcaa acccggtaat tttaacaaag acttctatga ctttgctgtg 14700
tctaagggtt tctttaagga aggaagttct gttgaattaa aacacttctt ctttgctcag 14760
gatggtaatg ctgctatcag cgattatgac tactatcgtt ataatctacc aacaatgtgt 14820
gatatcagac aactactacn tgtagttgaa gttgttgata agtactttga ttgttacgat 14880
ggtggctgta ttaatgctaa ccaagtcatc gtcaacaacc tagacaaatc agctggtttt 14940
ccattnnaca aatggggtaa ggctagactt tattatgatt caatgagtta tgaggatcaa 15000
gatgcacttt tcgcatatac aaaacgtaat gtcatcccta ctataactca aatgaatctt 15060
aagtatgcca ttagtgcaaa gaatagagct cgcaccgtag ctggtgtctc tatctgtagt 15120
actatgacca atagacagtt tcatcaaaaa ttattgaaat caatagccgc cactagagga 15180
gctactgtag taattggaac aagcaaattc tatggtggtt ggcacaacat gttaaaaact 15240
gtttatagtg atgtagaaaa ccctcacctt atgggttggg attatcctaa atgtgataga 15300
gccatgccta acatgcttag aattatggcc tcacttgttc ttgctcgcaa acatacaacn 15360
tgttgtagct tgtcacaccg tttctataga ttagctaatg agtgtgctca agtattgagt 15420
gaaatggtca tgtgtggcgg ttcactatat gttaaaccag gtggaacctc atcaggagat 15480
gccacaactg cttatgctaa tagtgttttt aacatttgtc aagctgtcac ggccaatgtt 15540
aatgcacttt tatctactga tggtaacaaa attgccgata agtatgtccg caatttacaa 15600
cacagacttt atgagtgtct ctaccnggat agagatgttg acacagactt tgtgaatgag 15660
ttttacgcat gtttgcgtaa acatttctca atgatgatac tctctgacga tgctgttgtg 15720
tgtttcaata gcacttatgc atctcaaggt ctagtggcta gcataaagaa ctttaagtca 15780
gttctttatt atcaaaatca tgtttttatg tctgaagcaa aatgttggac tgagactgac 15840
cttactaaag gacctcatga attttgctct caacatacaa tgctagttaa acagggtgat 15900
gattatgtgt accttcctta cccagatcca tcaagaatcc taggggccgg ctgttttgta 15960
gatgatatcg taaaaacaga tggtacactt atgattgaac ggttcgtgtc tttagctata 16020
gatgcttacc cacttactaa acatcctaat caggagtatg ctgatgtctt tcatttgtag 16080
cnacaataca taagaaagct acatgatgag ttaacaggac acatgttaga catgtattct 16140
gttatgctta ctaatgataa cacttcaagg tattgggaac ctgagtttta tgaggctatg 16200
cacacaccgc atacagtctt acaggctgtt ggggcttgtg ttctttgcaa ttcacagact 16260
tcattaagat gtggtgcttg catacgtaga ccattcttat gttgtaaatg ctgttacgac 16320
catgtcatat caacatcaca taaattagtc ttgtctgtta atccgtatgt ttgcaatgct 16380
ccaggttgtg atgtctgtga tgtgactcaa ctttacttag gaggtatgag ctattattgt 16440
aaatcacata aaccacccat tagttttcca ttgtgtgcta atggacaagt ttttggttta 16500
tataaaaata catgtgttgg tagcgataat gttactgact ttaatgcaat tgcaacatgt 16560
gactggacaa atgctggtga ttacatttta gctaacacct gtactgaaag actcaagctt 16620
tttgcagcag aaacgctcaa agctactgag gagacattta aactgtctta tggtattgct 16680
actgtacgtg aagtgctgac tgacagagaa ttacatcttt catgggaagt tggtaaacct 16740
agaccaccac ttaaccgaaa ttatgtcttt actggttatc gtgtaactaa aaacagtaaa 16800
gtacaaatag gagagtacac ctttgaaaaa ggtgactatg gtgatgctgt tgtttaccga 16860
ggtacaacaa cttacaaatt aaatgttggt gattattttg tgctgacatc acatacagta 16920
atgccattaa gtgcacctac actagtgcca caagagcact atgttagaat tactggctta 16980
tacccaacac tcaatatctc agatgagttt tctagcaatg ttgcaaatta tcaaaaggtt 17040
ggtatgcaaa agtattctac actccaggga ccacctggta ctggtaagag tcattttgct 17100
attggcctag ctctctacta cccttctgct cgcatagtgt atacagcttg ctctcatgcc 17160
gctgttgatg cactatgtga gaaggcatta aaatatttgc ctatagataa atgtagtaga 17220
attatacctg cacgtgctcg tgtagagtgt tttgataaat ccaaagtgaa ttcaacatta 17280
gaacagtatg tcttttgtac tgtaaatgca ttgcctgaga cgacagcaga tatagttgtc 17340
tttgatgaaa tttcaatggc cacaaattat gatttgagtg ttgtcaatgc cagattacgt 17400
gctaagcgct atgtgtacat tggcgaccct gctcaattac ctgcaccacg cacattgcta 17460
actaagggca cactagaacc agaatatttc aattcagtgt gtagacttat gaaaactata 17520
ggtccagaca tgttcgaagg aacttgtcgg cgttgtcctg ctgaaattgt tgacactgtg 17580
agtgctttgg tttatgataa taagcttaaa gcacataaag acaaatcagc tcaatgcttt 17640
aaaatgtttt ataagggtgt tatcacgcat gatgtttcat ctgcaattaa caggccacaa 17700
ataggcgtgg taagagaatt ccttacacgt aaccctgctt ggagaaaagc tgtctttatt 17760
tcaccttata attcacagaa tgctgtagcc tcaaagattt tgggactacc aactcaaact 17820
gttgattcat cacagggctc agaatatgcg nngcncatat tcactcaaac cactgaaaca 17880
gctcactctt gtaatgtaaa cagatttaat gttgctatta ccagagcaaa agtaggcata 17940
ctttgcataa tgtctgatag agacctttat gacaagttgc aatttacaag tcttgaaatt 18000
ccacgtagga atgtggcaac tttacaagct gaaaatgtaa caggactttg taaagattgt 18060
agtaaggtaa tcactgggtt acatcctaca caggcaccta cacacctcag tgttgccact 18120
aaattcaaaa ctgaaggttt atgtgttgac atacctggca tacctaagga catgacctat 18180
agaagactca tctccctgat gggttttaaa atgaattatc aagttaatgg ttaccctaac 18240
atgtttatca cccgcgaaga agctataaga catgtacgtg catggattgg cttcgatgtc 18300
gaggggtgtc atgctactag agaagctgtt ggtaccaatt tacctttaca gctaggtttt 18360
tctacaggng ttaacctagt tgctgtacct acaggttatg ttgatacacc taataataca 18420
gatttttcca gagttagtgc taaaccaccg cctggagatc aatttaaaca cctcatacca 18480
cttatgtaca aaggacttcc ttggaatgta gtgcgtataa agattgtaca aatgttaagt 18540
gacacactta aaaatctctc tgacagagtc gtatttgtct tatgggcaca tggctttgag 18600
ttgacatcta tgaagtattt tatgaaaata ggacctgagc gcacctgttg tctatgtgat 18660
agacgtgcca catgcttttc cactgcttca gacacttatg cctgatggca tcattctatt 18720
ggatttgatt acgtctataa tccgtttatg attgatgttc aacaatgggg ttttacaggt 18780
aacctacaaa gcaaccatga tctgtattgt caagtccatg gtaatgcaca tgtagctagt 18840
tgtgatgcaa tcatgactag gtgtctagct gtccacgagt gctttgttaa gcgtgttgac 18900
tggactattg aatatcctat aattggtgat gaactgaaga ttaatgcggc ttgtagaaag 18960
gttcaacaca tggttgttaa agctgcatta ttagcagaca aattcccagt tcttcacgac 19020
attggtaacc ctaaagctat taagtgtgta cctcaagctg atgtagaatg gaagttctat 19080
gatgcacagc cttgtagtga caaagcttat aaaatagann ncatnttcta ttcttatgcc 19140
acacattctg acaaattcac agatggtgta tgcctatttt ggaattgcaa tgtcgataga 19200
tatcctgcta attccattgt ttgtagattt gacactagag tgctatctaa ccttaacttg 19260
cctggttgtg atggtggcag tttgtatgta tataaacatg cattccacac accagctttt 19320
gataaaagtg cttttgttaa tttaaaacaa ttaccatttt tctattactc tgacagtcca 19380
tgtgagtctc atggaaaaca agtagtgtca gatatagatt atgtaccact aaagtctgct 19440
acgtgtatta cacgttgcaa tttaggtggt gctgtgtggt aacatcatgc taatgagtac 19500
agattgtatc tcgatgctta taacatgatg atctcagctg gctttagctt gtgggtttac 19560
aaacaatttg atacttataa cctctggaac acttttacaa gacttcagag tttagaaaat 19620
gtggctttta atgttgtaaa taagggacac tttgatggac aacagggtga agtaccagtt 19680
tctatcatta ataacactgt ttacacaaaa gttgatggtg ttgatgtaga attgtttgaa 19740
aataaaacaa cattgtgtgt taatgtagca tttgagcttt gggctaagcg caacattaaa 19800
ccagtaccag aggtgaaaat actcaataat ttgggtgtgg acattgctgc taatactgtg 19860
atctgggact acaaaagaga tgctccagca catatatcta ctattggtgt ttgttctatg 19920
actgacatag ccaagaaacc aactgaaacg atttgtgcac cactcactgt cttttttgat 19980
ggtagagttg atggtcaagt agacttattt agaaatgccc gtaatggtgt tcttattaca 20040
gaaggtagtg ttaaaggttt acaaccatct gtaggtccca aacaagctag tcttaatgga 20100
gtcacattaa ttggagaagc cgtaaaaaca cagttcaatt attataagaa agttgatggt 20160
gttgtccaac aattacctga aacttacttt actcagagta gaaatttaca agaatttaaa 20220
cccaggagtc aaatggaaat tgatttctta gaattagcta tggatgaatt cattgaacgg 20280
tataaattag aaaanttaac cttcgaacat atcgtttatg gagattttag tcatagtcag 20340
ttaggtggtt tacatctact gattggacta gctaaacgtt ttaaggaatc accttttgaa 20400
ttagaagatt ttattcctat ggacagtaca gttaaaaact atttcataac agatgcgcaa 20460
acaggttcat ctaagtgtgt gtgttctgtt gttgatttat tacttgatga ttttgttgaa 20520
ataataaaat nacangattt atctgtagtt tctaaggttg tcaaagtgac tattgactat 20580
acagaaattt catttatgct ttggtgtaaa gatggccatg tagaaacatt ttacccaaaa 20640
ttacaatcta gtcaagcgtg gcaaccgggt gttgctgtgc ctaatcttta caaaatgcaa 20700
agaatgctat tagaaaagtg tgaccttcaa aattatggtg atagtgcaac attacctaaa 20760
ggcataatga tgaatgtcgc aaaatatact caactgtgtc aatacttaaa cacattaaca 20820
ttagctgtac cctataatat gagagttata cattttggtg ctggttctga taaaggagtt 20880
gcaccaggta cagctgtttt aagacagtgg ttgcctacgg gtacgctgct tgtcgattca 20940
gatcttaatg actttgtctc tgatgcagat tcaactttga ttggtgattg tgcaactgta 21000
catacagcta ataaatggga tctcattatt agtgatatgt acgaccctaa gactaaaaat 21060
gttacaaaag aaaatgactc taaagagggt tttttcactt acatttgtgg gtttatacaa 21120
caaaaactag ctcttggagg ttccgtggct ataaagataa cagaacattc ttggaatgct 21180
gatctttaga agctcatggg acacttcgca tggtggacag cctttgttac taatgtgaat 21240
gcgtcatcat ctgaagcatt tttaattgga tgtaattatc ttggcaaacc acgcgaacaa 21300
atagatggtt atgtcatgca tgcaaattac atattttgga ggaatacaaa tccaattcag 21360
ttgtcttcct attctttatt tgacatgagt aaatttcccc tnantgttag gggtactgct 21420
gttatgtctt taaaagaagg tcaaatcaat gatatgattt tatctcttct tagtaaaggt 21480
agacttataa ttagagaaaa caactgattt gttatttcta gtgatgttct tgctaacaac 21540
taaacgaaca atgtttgttt ttcttgtttt attgccacta gtctctagtc agtgtgttaa 21600
tcttacaacc agaactcaat tacaccctgc atacactaat tctttcacac gtggtgttta 21660
ttaccctgac aaagttttca gatcctcagt tttacattca actcaggact tgttcttacc 21720
tttcttttcc aatgttactt ggttccatgc tatacatgtc tctgggacca atggtactaa 21780
gaggtttgat taccctgtcc taccatttga tgatggtgtt tattttgctt ccactgagaa 21840
gtctaacata ataagaggct ggatttttgg tactacttta gattcgaaga cccagtccct 21900
acttattgtt aataacgcta ctaatgttgt tattaaagtc tgtgaatttc aattttgtaa 21960
tgatccattt ttgggtgttt attaccacaa aaacaacaaa agttggatgg aaagtgagtt 22020
cagagtttat tctagtgcga ataattgcac ttttgaatat gtctctcagc cttttcttat 22080
ggaccttgaa ggaaaacagg gtaatttcaa aaatcttagg gaatttgtgt ttaagaatat 22140
tgatggttat tttaaaatat attctaagca cacgcctatt aatttagtgc gtgatctccc 22200
tcagggtttt tcggctttag aaccattggt agatttgcca ataggtatta acatcactag 22260
gtttcaaact ttacttgctt tacatagaag ttatttgact cctggtgatt cttcttcagg 22320
ttggacagct ggtgctgctg cttattatgt gggttatctt caacctagga cttttctatt 22380
aangtataat gaaaatggaa ccattacaga tgctgtagac tgtgcacttg accctctctc 22440
agaaacaaag tgtacgttga aatccttcac tgtagaaaaa ggaatctatc aaacttctaa 22500
ctttagagtc caaccaacag aatctgcnna tagatttcct aatattacaa acttgtgccc 22560
ttttggtgaa gtttttaacg ccaccagatt tgcatctgtt tatgcttgga acaggaagag 22620
aatcagcaac tgngttgctg attattctgt cctatataat tccgcatcat tttccacttt 22680
tcagtgttat ggagtgtctc ctactaaatt aaatgatctc tgctttacta atgtctatgc 22740
agattcattt gtaattagag gtgatgaagt cagacaaatc gctccagggc aaactggaaa 22800
gattgctgat tattattata aattaccaga tgattttaca ggctgcgtta tagcttggaa 22860
ttctaacaat cttgattcta aggttggtgg tgatgattcn tacctgtata gattgtttag 22920
gaagtctaat ctcaaacctt ttgagagaga tatttcaact gaaatctatc aggccggtag 22980
cacaccttgt aatggtgttg aaggttttaa ttgttacttt cctttacaat catatggttt 23040
ccaacccact aatggtgttg gtgaccaacc atacagagta gtagtacttt cttttgaact 23100
tctacatgca ccagcaactg tttgtggacc taaaaagtct actaatttgg ttaaaaacaa 23160
atgtgtcaat ttcaacttca atggtttaac aggcacaggt gttcttactg agtctaacaa 23220
aaagtttctg cctttccaac aatttggcag agacattgct gacactactg atgctgtccg 23280
aaacccacag acacttgaga ttcttgacat tacaccatgt tcttttggtg gtgtcagtga 23340
tgnggcacca ggaacaaata cttctaacca ggttgctgtt ctttatcagg atgttaactg 23400
cacagaagtc cctgttgcta ttcatgcaga tcaacttact cctacttggc gtgtttattc 23460
tacaggttct aatgtttttc aaacacgtgc aggctgttta ataggggctg aacatgtcaa 23520
caactcccag gagtgtgaca tacccattgg tgcaggtata tgcgctagtt atcagactca 23580
gactaattct cctcggcggg cacgtagtgt agctagtcaa tccatcattg cctacactat 23640
gtcacttggt gcagaaaatt cagttgctta ctctaataac tctattgcca tacccacaaa 23700
ttttactatt agtgttacca cagaaattct accagtgtct atgaccaaga catcagtaga 23760
ttgtagcnng tacatttgtg gtgattcaac tgaatgcagc aatcttttgt tgcaatatgg 23820
cagtttttgt acacaattaa accgtgcttt aactggaata gctgttgaac aagacaaaaa 23880
cacccaagaa gtttttgcac aagtcaaaca aatttacaaa acaccaccaa ttaaagattt 23940
tggtggtttt aatttttcac aaatattacc agatccatca aaaccaagca agaggtcatt 24000
tattgaagat ctacttttca acaaagtgac acttgcagat gctggcttca tcaaacaata 24060
tggtgattgc cttggtgata ttgctgctag agacctcatt tgtggacaaa agtttaacgg 24120
ccttactgtt ttgccacctt tgctcacaga tgaaatgatt gctcaataca cttctgcact 24180
gttagcgggt acaatcactt ctggttggac ctttggtgcg ggtgctgcat tacaaatnnc 24240
ntttgcanng ngcatggctt ataggtttaa tggtattgga gttacacaga atgttcagtc 24300
cgagaaccaa aaattgattg ccaaccaatt taatagtgct attggcaaaa ttcaagactc 24360
actttcttcc acagagngac attttggaaa acttcaagat gtggtcaacc aaaatgcaca 24420
agctttaaac acgcttgtta aacaacttag ctccaatttt ggtgcaattt caagtgtttt 24480
aaatgatatc ctttcacgtc ttgacaaagt tgaggctgaa gtgcaaattg ataggttgat 24540
cacaggcaga cttcaaagtt tgcagacata tgtgactcaa caattaatta gagctgcaga 24600
aatcagagct tctgctaatc ttgctgctac taaaatgtca gagtgtgtac ttggacaatc 24660
aaaaagagtt gatttttgtg gaaagggcta tcatcttatg tccttccctc agccagcacc 24720
tcatggtgta gtcttcttgc atgtgactta tgtccctgca caagaaaaga acttcacaac 24780
tgctcctgcc atttgtcatg atggaaaagc acactttcct cgtgaaggtg tctttgtttc 24840
aaatggcaca cactggtttg taacacaaag gaatttttat gaaccacaaa tcattactac 24900
agacaacaca tttgtgtctg gtaactgtga tgttgtaata ggaattgtca acaacacagt 24960
ttatgatcct ttgcaacctg aattagactc attcaaggag gagttagata aatattttaa 25020
gaatcacaca tcaccagatg ttgatttagg tgacatctct ggcattaatg cttcagttgt 25080
aaacattcaa aaagaaattg accgcctcaa tgaggttgcc aagaatttaa atgaatctct 25140
catcgatctc caagagcctg gaaagtatga gcagtatata aaatggccat ggtacatttg 25200
gctaggtttt atggctggct tgattgccat agtaatggtg acaattatgc tttgctgtat 25260
gaccagttgc tgtagttgtc tcaagggctg ttgttcttgt ggatcctgct gcaaatttga 25320
tgaagacgac tctgagccag tgctcaaagg agtcaaatta cattacacat aaacgaactt 25380
atggatttgt ttatgagaat cttcacaatt ggaactgtaa ctttgaagca aggtgaaatc 25440
aaggatgcta ctccttcaga ttttgttcgc gctactgcaa cgataccgat acaagcctca 25500
ctccctttcg gatggcttat tgttggcgtt gcacttcttg ctgtttttca gagcgcttcc 25560
aaaatcataa ccctcaaaaa gagatggcaa ctagcactct ccaagggtgt tcactttgtt 25620
tgcgacttgc tgttgttgtt tgtaacagtt tactcacacc ttttgctcgt tgctgctggc 25680
cttgaagccc cttttctcta tctttatgct ttagtctact tcttgcagag tataaacttt 25740
gtaagaataa taatgaggct ttggctttgc tggaaatgcc gttccaaaaa cccattactt 25800
tatgatgcca actattttct ttgctggcat actaattgtt acgactattg tataanttac 25860
aatagtgtaa cttcttcaat tgtcattact tcaggtgatg gcacaacaag tcctatttct 25920
gaacatgact accagatggg tggttatact gaaaaatggg aatctggagt aaaagactgt 25980
gttgtgngac acagttactt cacttcagac tattaccagc tgtactcaac tctatngagt 26040
acagacactg gtgttgaaca tgttaccttc ttcatctaca cnnagattgt tgatgagcct 26100
gaagaacatg tccaaattca cacaatcgac ggttcatccg gagttgttaa tccagtaatg 26160
gaaccaattt atgatgaacc gacgacgact actagcgtgc ctttgtaanc acaagctgat 26220
gagtacgaac ttatgtactc attcgtttcg ggagagacag gtacgttaat agttaatagc 26280
gtacttcttt ttcttgcttt cgtggtattc ttgctagtta cactagccat ccttactgcg 26340
cttcgattgt gtgcgtactg ctgcaatatt gttaacgtga gtcttgtaaa accttctttt 26400
tacgtttact ctcgtgttaa aaatctgaat tcttctagag ttcctgatct tctggtctaa 26460
acgaactaaa tgntanatta gtttttctgt ttggaacttt aattttagcc ttggcagatt 26520
ccaacggtac atgtaccgtt gaagagctna gcaagctcct tgaacaatgg aacctagcnn 26580
cnnnattcct attccttaca tggatttgtc ttctacaatt tgcctatgcc aacaggaata 26640
ggtttttgta tataattaag ttaattttcc tctggctgtt atggccagta actttagctt 26700
gttttgtgct tgctgctgtt tacagaataa attggatcac cggtggaatt gctatcgcaa 26760
tggcttgtct tgtaggcttg atgtggctca gctacttcat tgcttctttc agactgtttg 26820
cgcgtacgcg ttccatgtgg tcattcaatc cagaaactaa cattcttctc aacgtgccac 26880
tccatggcac tattctgacc agaccgcttc tagaaagtga actcgtaatc ggagctgtga 26940
tccttcgtgg acatcttcgt attgctggac accatctagg acgctgtgac atcaaggacc 27000
tgcctaaaga aatcactgtt gctacatcac gaacgctttc ttattacaaa ttgggagctt 27060
cgcagcgtgt agcaggtgac tcaggttttg ctgcatacag tcgctacagg attggcaact 27120
ataaattaaa cacagaccat tccagtagca gtgacaatat tgctttgctt gtacagtaag 27180
tgacaacaga tgtttcatct cgttgacttt caggttacta tagcagagat attactaatt 27240
attatgagga cttttaaagt ttccatttgg aatcttgatt acatcataaa cctcataatt 27300
aaaaatttat ctaagtcact aactgagaan nacanttctc aattagatga agagcaacca 27360
atggagattg attaaacgaa catgaaaatt attcttttct tggcactgat aacactcgct 27420
acttgtgagc tttatcacta ccaagagtgt gttagaggta caacagtact tttaaaagaa 27480
ccttgctctt ctggaacata cgagggcaat tcaccatttc atcctctagc tgataacaaa 27540
tttgcactga cttgctttag cactcaattt gcttttgctt gtcctgacgg cgtaaaacac 27600
gtctatcagt tacgtgccag atcagtttca cctaaactgt tcatcagaca agaggaagtt 27660
cangaacttt actctccaat ttttcttatt gttgcggcaa tagtgttncn aacactttgc 27720
ttcacactca aaagaaagac agaatgattg aactttcatt aattgacttc tatttgtgct 27780
ttttagcctt tctgctattc cttgttttaa ttatgcttat tatcttttgg ttctcacttg 27840
aactgcaaga tcataatgaa acttgtcacg cctaaacgaa catgaaattt cttgttttct 27900
taggaatcat cacaactgta gctgcatttc accaagaatg tagtttacag tcatgtactc 27960
aacatcaacc atatgtagtt gatgacccgt gtcctattca cttctattct aaatggtata 28020
ttagagtagg agctagaaaa tcagcacctt taattgaatt gtgcgtggat gaggctggtt 28080
ctaaatcacc cattcagtac atcgatatcg gtaattatac agtttcctgt tcacctttta 28140
caattaattg ccaggaacct aaattgggta gtcttgtagt gcgttgttcg ttctatgaag 28200
actttttaga gtatcatgac gttcgtgttg ttttagattt aatctaaacg aacaaactaa 28260
aatgtctgat aatggacccc aaaatcagcg aaatgcaccc cgcattacgt ttggtggacc 28320
ctcagattca actggcagta accagaatgg agaacgcagt ggggcgcgat caaaacaacg 28380
tcggccccaa ggtttaccca ataatactgc gtcttggttc accgctctca ctcaacatgg 28440
caaggaagac cttaaattcc ctcgaggaca aggcgttcca attaacacca atagcagtcc 28500
agatgaccaa attggctact accgaagagc taccagacga attcgtggtg gtgacggtaa 28560
aatgaaagat ctcagtccaa gatggtattt ctactaccta ggaactgggc cagaagctgg 28620
acttccctat ggtgctaaca aagacggcat catatgggtt gcaactgagg gagccttgaa 28680
tacaccaaaa gatcacattg gcacccgcaa tcctgctaac aatgctgcaa tcgtgctaca 28740
acttcctcaa ggaacaacat tgccaaaagg cttctacgca gaagggagca gaggcggcag 28800
tcaagcctct tctcgttcct catcacgtag tcgcaacagt tcaagaaatt caactccagg 28860
cagcagtagg ggaacttctc ctgctagaat ggctggcaat ggcggtgatg ctgctcttgc 28920
tttgctgctg cttgacagat tgaaccagct tgagagcaaa atgtctggta aaggccaaca 28980
acaacaaggc caaactgtca ctaagaaatc tgctgctgag gcttcgagca agcctcggca 29040
aaaacgtact gccactaaag catacaatgt aacacaagct ttcggcagac gtggtccaga 29100
acaaacccaa ggaaattttg gggaccagga actaatcaga caaggaactg attacaaaca 29160
ttggccgcaa attgcacaat ttgcccccag cgcttcagcg ttcttcggaa tgtcgcgcat 29220
tggcatggaa gtcacacctt cgggaacgtg gttgacctac acaggtgcca tcaaattgga 29280
tgacaaagat ccaaatttca aagatcaagt cattttgctg aataagcata ttgacgcata 29340
caaaacattc ccaccaacag agcctaaaaa ggacaaaaag aagaaggctg atgaaactca 29400
agccttaccg cagagacaga agaaacagca aactgtgact cttcttcctg ctgccgattt 29460
ggatgatttc tccaaacaat tgcaacaatc catgagcagt gctgactcaa ctcaggccta 29520
aactcatgca gaccacacaa ggcagatggg ctatataaac gttttcgctt ttccgtttac 29580
gatatatagt ctactcttgt gcagaatgaa ttctcgtaac tacatagcac aagtagatgt 29640
agttaacttt aatctcacat agcaatcttt aatcagtgtg taacattagg gaggacttga 29700
aagagccacc acattttcac cgaggccacg cggagtacga tcgagtgtac agtgaacaat 29760
gctagggaga gctgcctata tggaagagcc ctaatgtgta aaattgattt tagtagtgct 29820
atccccctct gattttnatg gccatg 29846
Claims (6)
1. A gene chip capable of being used for detecting the presence or absence of a novel coronavirus SARS-CoV-2 and, if present, determining the genomic sequence of SARS-CoV-2, said gene chip comprising 29846 sets of probes, each set of probes comprising 8 probes of 25bp in length, the 8 probes covering the same segment of the SARS-CoV-2 genome, the first probe in each set of probes being identical to the segment of the SARS-CoV-2 genome, the second, third and fourth probes being different from the first probe only at one specific site and being the remaining three bases in A, T, C, G at the specific site, the fifth, sixth, seventh and eighth probes being fully complementary to the first, second, third and fourth probes, respectively, the specific sites in each set of probes correspond to different sites in the SARS-CoV-2 genome and all the specific sites completely cover the 13 th-29858 th of the SARS-CoV-2 genome, and the specific site is the 13 th site in the probes.
2. The gene chip of claim 1, wherein the gene chip is an in situ synthesis gene chip.
3. A kit comprising the gene chip according to claim 1 or 2.
4. A method which can be used to detect the presence of the novel coronavirus SARS-CoV-2 and, if present, to determine the genomic sequence of SARS-CoV-2, said method comprising the steps of:
(1) carrying out RNA extraction, reverse transcription, amplification, fluorescence labeling and fragmentation on an experimental sample to be detected;
(2) hybridizing the product of step (1) with the gene chip according to claim 1 or 2,
(3) determining whether the novel coronavirus SARS-CoV-2 exists in the experimental sample to be detected based on the hybridization signal of the probe on the gene chip and the product of the step (1) and determining the genome sequence of SARS-CoV-2 if the novel coronavirus SARS-CoV-2 exists.
5. The method of claim 4, wherein the product of step (1) has a length of 30-70bp, preferably 40-60bp, more preferably 45-55 bp.
6. Use of the gene chip according to claim 1 or 2 or the kit according to claim 3 or the method according to claim 4 or 5 for the early warning of new viruses within the subfamily coronaviruses.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010981983.7A CN111961763A (en) | 2020-09-17 | 2020-09-17 | Novel gene chip for detecting coronavirus |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010981983.7A CN111961763A (en) | 2020-09-17 | 2020-09-17 | Novel gene chip for detecting coronavirus |
Publications (1)
| Publication Number | Publication Date |
|---|---|
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| CN113186341A (en) * | 2021-03-30 | 2021-07-30 | 贵州省疾病预防控制中心 | CRISPR-mediated one-step constant-temperature amplification SARS-CoV-2 detection method |
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| CN113186341A (en) * | 2021-03-30 | 2021-07-30 | 贵州省疾病预防控制中心 | CRISPR-mediated one-step constant-temperature amplification SARS-CoV-2 detection method |
| CN113186341B (en) * | 2021-03-30 | 2022-11-22 | 贵州省疾病预防控制中心 | CRISPR-mediated one-step constant-temperature amplification SARS-CoV-2 detection method |
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