Kit for detecting novel coronavirus and detection method

Abstract

The invention provides a kit for detecting novel coronavirus and a detection method, relates to the technical field of gene detection, and comprises a specific primer probe combination suitable for a method of combining swab direct amplification with fluorescent PCR (Polymerase Chain Reaction), and a kit for detecting an N gene and an O gene in the novel coronavirus by using the combination. According to the kit for detecting the novel coronavirus and the detection method, the sample size demand is low, the steps of extracting and purifying nucleic acid substances are saved, meanwhile, the amplification reaction speed is high, a report can be issued within 17 minutes at the soonest, the method is simple, convenient, rapid and accurate, the cost is saved, and the method does not need blood collection and is easy to operate. And during clinical application, the inconvenience of collecting the nasopharyngeal swab by a patient is reduced, and the waste of medical instrument resources is also reduced.

Description

Technical Field

[0001] The present invention relates to the field of gene detection technology, and in particular to a kit and a method for detecting a novel coronavirus. Background Art

[0002] Currently, most methods used for COVID-19 nucleic acid detection are based on two methods: RT-PCR amplification and isothermal amplification. The COVID-19 nucleic acid detection reagents used in medical laboratories are mainly real-time fluorescence RT-PCR. Among them, the real-time fluorescence RT-PCR reagents undergo specimen pretreatment, nucleic acid extraction and purification, and then amplification and product analysis are completed on a real-time fluorescence RT-PCR instrument. The isothermal amplification reagents also undergo specimen pretreatment, nucleic acid extraction and purification, and then amplification and product analysis are completed on an isothermal amplifier or analyzer, or product analysis is completed through methods such as CRISPR.

[0003] Isothermal amplification is usually claimed to be faster and is sometimes also called "rapid nucleic acid detection reagent". However, when the specimen pretreatment, nucleic acid extraction and purification are the same as real-time fluorescence RT-PCR, the shortening of nucleic acid amplification and product analysis time does not have a very obvious impact on the overall detection time. In particular, when the number of specimens to be tested is large, increasing the number of real-time fluorescence RT-PCR instruments can also increase the detection throughput, thereby completing the same number of specimen tests in the same time. However, whether it is PCR or isothermal amplification, it needs to be carried out in a clinical gene amplification detection laboratory with laboratory partitioning and anti-contamination procedures. At present, the most commonly used methods on the market are mostly to extract and purify nucleic acid substances (DNA) based on nasal swabs and oral swabs, and then test them. The PCR method based on DNA extraction has many experimental steps, a long process, and is prone to contamination during the detection process. In response to the above-mentioned drawbacks, this paper proposes a field timely detection method and kit with comprehensive detection sites, convenient and fast operation. Summary of the invention

[0004] The purpose of the present invention is to provide a kit and a method for detecting the new coronavirus to solve the above-mentioned technical problems.

[0005] In order to solve the above technical problems, the present invention adopts the following technical solutions: A specific primer-probe combination suitable for swab direct amplification combined with fluorescent PCR method, wherein the target genes for the new coronavirus are N gene and O gene respectively, and the specific primer-probe combination includes upstream and downstream primers of the target gene and a fluorescent probe.

[0006] The N gene specific primer probe combination comprises an upstream primer having a nucleotide sequence as shown in SEQ ID NO.1, a downstream primer having a nucleotide sequence as shown in SEQ ID NO.2, and a fluorescent probe having a nucleotide sequence as shown in SEQ ID NO.3, wherein FAM is a fluorescent modification group and MGB is a fluorescent quenching group; The O gene-specific primer-probe combination comprises an upstream primer having a nucleotide sequence as shown in SEQ ID NO.4, a downstream primer having a nucleotide sequence as shown in SEQ ID NO.5, and a fluorescent probe having a nucleotide sequence as shown in SEQ ID NO.6, wherein VIC is a fluorescent modification group and MGB is a fluorescent quenching group.

[0007] The application of the specific primer-probe combination provided by the present invention in the preparation of products for detecting the novel coronavirus is to detect products for detecting the novel coronavirus.

[0008] This product is a kit for detecting the N gene and O gene in the new coronavirus. The kit includes a specific primer probe combination, a primer probe combination of an internal reference gene, an amplification enzyme, and ultrapure water.

[0009] It should be noted that the fluorescent probe in the primer-probe combination of the internal reference gene and the fluorescent probe in the specific primer-probe combination use different fluorescent modification groups (ROX modification); The primer-probe combination of the internal reference gene comprises an upstream primer with a nucleotide sequence as shown in SEQ ID NO.7, a downstream primer with a nucleotide sequence as shown in SEQ ID NO.8, and a fluorescent probe with a nucleotide sequence as shown in SEQ ID NO.9, wherein ROX is a fluorescent modification group and MGB is a fluorescent quenching group.

[0010] Preferably, the final concentration of the upstream and downstream primers for amplifying the N gene is 0.2 μmol / L, and the final concentration of the probe for detecting the N gene is 0.1 μmol / L.

[0011] Preferably, the final concentration of the upstream and downstream primers for amplifying the O gene is 0.4 μmol / L, and the final concentration of the probe for detecting the O gene is 0.05 μmol / L.

[0012] Preferably, the final concentration of the upstream and downstream primers of the primer pair for amplifying the internal reference gene is 0.05 μmol / L, and the final concentration of the probe for detecting the internal reference gene is 0.025 μmol / L.

[0013] The kit also includes a positive quality control product and a negative quality control product, wherein the positive quality control product is a pseudovirus of the target amplified fragment and the internal standard fragment, and the negative quality control product is a pseudovirus containing the internal standard fragment.

[0014] A method for detecting a novel coronavirus for non-diagnostic purposes, comprising the following steps: 1) Obtain the oral swab or nasal swab sample to be tested and place it in ultrapure water to make swab liquid as an amplification template; 2) preparing a PCR reaction system including an N gene upstream primer, an N gene downstream primer, an N gene fluorescent probe, an O gene upstream primer, an O gene downstream primer, an O gene fluorescent probe, an internal reference upstream primer, an internal reference downstream primer, an internal reference fluorescent probe, an amplification enzyme and ultrapure water, and mixing the reaction system thoroughly and lyophilizing it; 3) Dissolve the positive control and negative control separately with enzyme-free water, transfer the solution to the lyophilized reagent prepared in step 2) for dissolution and transfer to the corresponding reaction tube for amplification; 4) Add the oral swab or nasal swab sample obtained in step 1) to the freeze-dried reagent in step 2), run the real-time fluorescence PCR amplification program, and determine whether the sample contains the new coronavirus gene based on the Ct value.

[0015] The beneficial effects of the present invention are: The swab direct amplification combined with fluorescent PCR method proposed in the present invention requires a small amount of sample, saves the extraction and purification steps of nucleic acid substances, and at the same time, the amplification reaction speed of the present invention is fast, and the fastest report can be issued in only 17 minutes. It is simple, fast, accurate and cost-saving. This method does not require blood collection. When used clinically, it reduces the inconvenience of patients collecting nasopharyngeal swabs and reduces the waste of medical device resources. DETAILED DESCRIPTION

[0016] In order to facilitate the understanding of the present invention, the present invention is described more fully below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0017] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0018] Example 1 In this embodiment, a specific primer-probe combination of a swab direct amplification combined with a fluorescent PCR method is proposed. The target genes of the new coronavirus are the N gene and the O gene, respectively. The specific primer-probe combination includes upstream and downstream primers of the target gene and a fluorescent probe.

[0019] The N gene specific primer probe combination comprises an upstream primer having a nucleotide sequence as shown in SEQ ID NO.1, a downstream primer having a nucleotide sequence as shown in SEQ ID NO.2, and a fluorescent probe having a nucleotide sequence as shown in SEQ ID NO.3, wherein FAM is a fluorescent modification group and MGB is a fluorescent quenching group; The O gene-specific primer-probe combination comprises an upstream primer having a nucleotide sequence as shown in SEQ ID NO.4, a downstream primer having a nucleotide sequence as shown in SEQ ID NO.5, and a fluorescent probe having a nucleotide sequence as shown in SEQ ID NO.6, wherein VIC is a fluorescent modification group and MGB is a fluorescent quenching group.

[0020] Example 2 This embodiment proposes a kit for detecting the N and O genes of the new coronavirus, including the specific primer probe combination in Example 1, the primer probe combination of the internal reference gene, an amplification enzyme and ultrapure water.

[0021] It is further explained that the fluorescent probe in the primer-probe combination of the internal reference gene and the fluorescent probe in the specific primer-probe combination use different fluorescent modification groups (ROX modification).

[0022] Further, the primer-probe combination of the internal reference gene includes an upstream primer with a nucleotide sequence as shown in SEQ ID NO.7, a downstream primer with a nucleotide sequence as shown in SEQ ID NO.8, and a fluorescent probe with a nucleotide sequence as shown in SEQ ID NO.9, ROX is a fluorescent modification group, and MGB is a fluorescent quenching group.

[0023] The final concentration of the upstream and downstream primers for amplifying the N gene was 0.2 μmol / L, and the final concentration of the probe for detecting the N gene was 0.1 μmol / L.

[0024] The final concentration of the upstream and downstream primers for amplifying the O gene was 0.4 μmol / L, and the final concentration of the probe for detecting the O gene was 0.05 μmol / L.

[0025] The final concentration of the upstream and downstream primers of the primer pair for amplifying the internal reference gene was 0.05 μmol / L, and the final concentration of the probe for detecting the internal reference gene was 0.025 μmol / L.

[0026] The test kit for detecting the N and O genes of the new coronavirus also includes positive quality control products and negative quality control products. The positive quality control product is a pseudovirus with the target amplification fragment and the internal standard fragment, and the negative quality control product is a pseudovirus containing the internal standard fragment.

[0027] Example 3 This embodiment proposes a swab direct amplification combined with fluorescent PCR detection method for novel coronavirus gene detection, which includes the following steps: 1) Obtain the oral swab or nasal swab sample to be tested and place it in 200 μL ultrapure water to make swab liquid; 2) Prepare the PCR reaction system, which includes N gene upstream primer, N gene downstream primer, N gene fluorescent probe, O gene upstream primer, O gene downstream primer, O gene fluorescent probe, internal reference upstream primer, internal reference downstream primer, internal reference fluorescent probe, amplification enzyme and ultrapure water. The reaction system should be fully mixed. The 25 μL reaction system is shown in Table 1: Table 1: Composition of the reaction system ; 3) The reaction system mixed in step 2) is freeze-dried. The freeze-drying procedure is shown in Table 2: Table 2: Lyophilization schedule for the reaction system ; 4) Dissolve the positive control and negative control products with 25 μL enzyme-free water respectively, and then transfer the solution to the freeze-dried reagent configured in step 3) for dissolution, and transfer to the corresponding reaction tube for amplification. It should be noted that if the amplification curve of the positive control product configured in step 4) for the target / internal standard channel (FAM, VIC, ROX) is S-shaped, and the Ct value is ≤40, the reagent is considered qualified, otherwise it needs to be re-amplified; if the Ct value of the target channel (FAM, VIC) in the negative control tube is 0 or >40, and the amplification curve of the internal standard channel (ROX) is S-shaped, and the Ct value is ≤40, the reagent is considered to be uncontaminated and the reagent result is credible; 5) Add 25 μL of the swab sample obtained in step 1) to the freeze-dried reagent in step 3), run the real-time fluorescence PCR amplification program, and determine whether the sample has the new coronavirus gene based on the Ct value. The judgment criteria are shown in Table 3: Table 3: Ct value interpretation table of novel coronavirus genes ; It should be noted that in step 4), the real-time fluorescence PCR amplification program was run as follows: reverse transcription at 50°C for 5 min, pre-denaturation at 95°C for 30 s; denaturation at 95°C for 3 s, annealing at 69°C for 15 s, for a total of 45 cycles.

[0028] The swab direct amplification combined with fluorescent PCR detection method for novel coronavirus gene detection proposed in this embodiment requires less sample volume (only 25ul swab mixture is required), saving the extraction and purification steps of nucleic acid substances. At the same time, the amplification reaction speed of the present invention is fast, and the report can be issued in as little as 17 minutes. It is simple, fast, accurate and cost-saving. At present, the POCT market is developing steadily worldwide. This method does not require blood collection. When used clinically, it reduces the inconvenience of patients collecting nasopharyngeal swabs and reduces the waste of medical device resources.

[0029] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0030] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A specific primer-probe combination for detecting a new coronavirus, characterized in that: The specific primer-probe combination targets the N gene and O gene in the target gene of the new coronavirus. The specific primer-probe combination includes upstream and downstream primers of the target gene and a fluorescent probe; The N gene specific primer probe combination comprises an upstream primer having a nucleotide sequence as shown in SEQ ID NO.1, a downstream primer having a nucleotide sequence as shown in SEQ ID NO.2, and a fluorescent probe having a nucleotide sequence as shown in SEQ ID NO.3, wherein FAM is a fluorescent modification group and MGB is a fluorescent quenching group; The O gene-specific primer-probe combination comprises an upstream primer having a nucleotide sequence as shown in SEQ ID NO.4, a downstream primer having a nucleotide sequence as shown in SEQ ID NO.5, and a fluorescent probe having a nucleotide sequence as shown in SEQ ID NO.6, wherein VIC is a fluorescent modification group and MGB is a fluorescent quenching group.

2. Application of the specific primer-probe combination described in claim 1 in the preparation of a product for detecting a new coronavirus.

3. The use according to claim 2, characterized in that: The product for detecting the new coronavirus is a kit for detecting the N gene and O gene in the new coronavirus.

4. A kit for detecting N gene and O gene in a novel coronavirus, characterized in that: It comprises a specific primer probe combination, an internal reference gene primer probe combination, an amplification enzyme and ultrapure water, wherein the fluorescent probe in the internal reference gene primer probe combination and the fluorescent probe in the specific primer probe combination adopt different fluorescent modification groups.

5. The kit for detecting N gene and O gene in novel coronavirus according to claim 4, characterized in that: The primer-probe combination of the internal reference gene comprises an upstream primer with a nucleotide sequence as shown in SEQ ID NO.7, a downstream primer with a nucleotide sequence as shown in SEQ ID NO.8, and a fluorescent probe with a nucleotide sequence as shown in SEQ ID NO.9, wherein ROX is a fluorescent modification group and MGB is a fluorescent quenching group.

6. The kit for detecting N gene and O gene in novel coronavirus according to claim 5, characterized in that: The final concentration of the upstream and downstream primers for amplifying the N gene is 0.2 μmol / L, and the final concentration of the probe for detecting the N gene is 0.1 μmol / L; The final concentration of the upstream and downstream primers for amplifying the O gene was 0.4 μmol / L, and the final concentration of the probe for detecting the O gene was 0.05 μmol / L; The final concentration of the upstream and downstream primers of the primer pair for amplifying the internal reference gene was 0.05 μmol / L, and the final concentration of the probe for detecting the internal reference gene was 0.025 μmol / L.

7. The kit for detecting N gene and O gene in novel coronavirus according to claim 5, characterized in that: Also includes positive and negative quality controls.

8. The kit for detecting N gene and O gene in novel coronavirus according to claim 7, characterized in that: The positive quality control product is a pseudovirus containing the target amplified fragment and the internal standard fragment, and the negative quality control product is a pseudovirus containing the internal standard fragment.

9. The kit for detecting N gene and O gene in novel coronavirus according to claim 8, characterized in that: The liquid reagents in the kit are made into freeze-dried powder by an in-situ freeze-drying machine.

10. A method for detecting a novel coronavirus for non-diagnostic purposes, characterized in that: The steps include: 1) Obtain the oral swab or nasal swab sample to be tested and place it in ultrapure water to make swab liquid as an amplification template; 2) preparing a PCR reaction system including an N gene upstream primer, an N gene downstream primer, an N gene fluorescent probe, an O gene upstream primer, an O gene downstream primer, an O gene fluorescent probe, an internal reference upstream primer, an internal reference downstream primer, an internal reference fluorescent probe, an amplification enzyme and ultrapure water, and mixing the reaction system thoroughly and lyophilizing it; 3) Dissolve the positive control and negative control separately with enzyme-free water, transfer the solution to the lyophilized reagent prepared in step 2) for dissolution and transfer to the corresponding reaction tube for amplification; 4) Add the oral swab or nasal swab sample obtained in step 1) to the freeze-dried reagent in step 2), run the real-time fluorescence PCR amplification program, and determine whether the sample contains the new coronavirus gene based on the Ct value.