CN112795703A

CN112795703A - Primer, probe and kit for detecting SARS-CoV-2

Info

Publication number: CN112795703A
Application number: CN202110254686.7A
Authority: CN
Inventors: 夏欣一; 吴秋月; 李卫巍; 罗涛
Original assignee: Eastern Theater General Hospital of PLA
Current assignee: Eastern Theater General Hospital of PLA
Priority date: 2021-03-09
Filing date: 2021-03-09
Publication date: 2021-05-14

Abstract

The invention relates to the field of biotechnology, in particular to primers and probes for identifying novel coronavirus (SARS-CoV-2) and a kit thereof. The primer probe combination provided by the invention comprises a primer and a probe aiming at SARS-CoV-2. The primer and the probe have good specificity, and can realize the rapid, accurate and sensitive identification of SARS-CoV-2 by combining with a real time PCR detection method. Experiments show that the lowest detection limit of SARS-CoV-2 by the primer probe of the invention is 20 copies/ml.

Description

Primer, probe and kit for detecting SARS-CoV-2

Technical Field

The invention relates to the field of biotechnology, in particular to a primer probe combination for identifying SARS-CoV-2.

Background

The novel coronavirus was named 2019-nCoV by the world health organization at 12.1.2020 and SARS-CoV-2 by the International Committee for Classification of viruses at 11.2.2020.

SARS-CoV-2 has extremely strong infectivity, is mainly transmitted (sneezing and coughing) through respiratory droplets and contagious infection, mainly causes respiratory system infection of human beings, and infected persons often have symptoms of fever, hypodynamia, dry cough, gradually dyspnea and the like, most patients have good prognosis, some patients have slight illness symptoms and no obvious fever, severe persons have symptoms of acute respiratory distress syndrome, septic shock, metabolic acidosis which is difficult to correct, blood coagulation dysfunction and the like, and critically ill persons can cause death. SARS-CoV-2 is the 7 th coronavirus reported to infect humans, the remaining 6 are HCoV-229E, HCoV-OC43, SARS-CoV, HCoV-NL63, HCoV-HKU1, and MERS-HCoV, respectively. Wherein HCoV-229E and HCoV-NL63 belong to the genus alpha coronavirus, and HCoV-OC43, SARS-CoV, HCoV-HKU1, MERS-CoV and 2019-nCoV are all the genus beta coronavirus.

Nucleic acid detection is a common method for clinical etiology diagnosis, and has the advantages of high sensitivity, strong specificity and the like. Since the epidemic situation of SARS-CoV-2 pneumonia occurs, nucleic acid detection is an important diagnostic basis for clinical diagnosis, isolation release and recovery discharge. The real-time fluorescence PCR technology based on the TaqMan probe method has the advantages of simplicity, rapidness and accuracy, is widely applied to detection of clinical pathogens, makes an important contribution to epidemic situation control of SRAS viruses outbreaked in China in 2003, is one of MERS-CoV virus detection methods recommended by WHO, and is listed in 2019-nCoV diagnosis and treatment guidelines in China at present. Corresponding detection schemes, including primer sequences, are also published by the Chinese centers for disease control, the U.S. CDC and other units. In the global influenza shared database (GISAID), the whole genome sequence of 2019-nCoV exceeds three thousand, and the published primer sequences have more or less primer region mutations through sequence analysis.

Disclosure of Invention

In view of the above, the technical problem to be solved by the present invention is to provide a primer-probe combination for SARS-CoV-2 identification, which can accurately, rapidly and sensitively realize the identification of SARS-CoV-2.

The primer probe combination provided by the invention comprises:

the primer probe combination comprises the following components:

the upstream primer of SARS-CoV-2N gene shown in SEQ ID NO. 1;

the downstream primer of SARS-CoV-2N gene shown in SEQ ID NO. 2;

a specific probe of SARS-CoV-2N gene as shown in SEQ ID NO. 3;

the upstream primer of SARS-CoV-2ORF1ab gene shown in SEQ ID NO. 4;

the downstream primer of SARS-CoV-2ORF1ab gene shown in SEQ ID NO. 5;

and a specific probe of SARS-CoV-2ORF1ab gene as shown in SEQ ID NO. 6.

The primer and probe of SARS-CoV-2 in the invention are used for qualitative detection of SARS-CoV-2.

In the invention, the amplified fragment of the primer pair shown in SEQ ID NO. 1-2 is shown in SEQ ID NO. 7;

the fragments amplified by the primer pairs shown as SEQ ID NO. 4-5 are shown as SEQ ID NO. 8.

In the invention, the 3 'end of the probe is connected with a quenching group, and the 5' end of the probe is connected with a fluorescent group; wherein:

the 5' end of the probe shown as SEQ ID NO.3 is connected with FAM fluorophore;

the 5' end of the probe shown as SEQ ID NO.6 is connected with ROX fluorescent group.

The primer probe combination of the invention is applied to the preparation of a kit for detecting SARS-CoV-2.

The primer and the probe provided by the invention can simultaneously detect two targets of SARS-CoV-2 in one reaction system, have good accuracy, specificity and sensitivity, and can save cost. Because the invention adopts the probe with higher specificity to be applied to the kit, the SARS-CoV-2 nucleic acid in an unknown sample can be rapidly detected, reliable experimental basis is provided for diagnosing SARS-CoV-2, and the technical problems of low efficiency, poor specificity and low sensitivity of the existing kit are solved.

The invention also provides a nucleic acid detection kit for SARS-CoV-2, which comprises the primer probe combination and Real time PCR reaction reagent.

The Real time PCR reaction reagent comprises: dNTPs, M-MLV enzyme, Taq enzyme, MgCl₂。

Wherein the concentration of dNTPs is 10mM, M-MLV enzyme concentration of 200u/ul, Taq enzyme concentration of 5u/ul, MgCl₂Is 50 mM.

The kit also comprises a negative control and a positive control; wherein the negative control is sterile water, and the positive control is artificially synthesized with a concentration of 1 × 10⁶Pseudoviruses of Copies/ml.

The invention also provides a method for detecting SARS-CoV-2 for non-diagnostic or therapeutic purposes: the primer probe combination is adopted to detect Real time PCR of the sample, and whether the sample is infected by SARS-CoV-2 is judged according to the Ct value of the detection.

The judgment comprises the following steps:

the channel CT values shown in SEQ ID NO.3 and SEQ ID NO.6 are more than 40 or no CT value, and the reported detection result is negative;

the CT value of any one or two of the two probe channels shown in SEQ ID NO.3 and SEQ ID NO.6 is less than or equal to 40, and the result is reported to be positive for SARS-CoV-2.

The non-diagnostic detection reagent mainly comprises:

the reaction program of the Real time PCR comprises the following steps:

has the advantages that:

the primer probe combination provided by the invention comprises a primer and a probe aiming at SARS-CoV-2. The primer and the probe have good specificity, and can realize the rapid, accurate and sensitive identification of SARS-CoV-2 by combining with a real time PCR detection method. Experiments show that the minimum detection limit of the primer probe for detecting SARS-CoV-2 can reach 20copies/ml, the amplification efficiency of the primer probe designed by the invention aiming at an amplification sequence is higher, the required denaturation and annealing time is shorter, and the total reaction time is 60-70 min. The lowest detection limit of other PCR detection kits of the same type is usually 50-1000copies/ml, and the reaction time is usually more than 80 min.

Drawings

FIG. 1 shows the confirmation of the minimum detection limit of the N gene by the kit of the present invention

FIG. 2 is the confirmation of the comparison kit against the minimum detection limit of the N gene

FIG. 3 shows the confirmation of the minimum detection limit of ORF1ab gene by the kit of the present invention

FIG. 4 is the confirmation of the lowest detection limit of the comparison kit for ORF1ab gene

FIG. 5 shows the confirmation of the minimum detection limit for the N gene by the comparative example kit of the present invention

FIG. 6 is a view showing confirmation of the minimum detection limit of ORF1ab gene by the comparative example kit of the present invention

Detailed Description

The invention provides a primer probe combination for identifying SARS-CoV-2, and the technical personnel can use the content to reference the text and properly improve the technological parameters to realize the method. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.

The test materials and instruments adopted by the invention are all common commercial products and can be purchased in the market.

The invention is further illustrated by the following examples:

EXAMPLE 1 preparation of SARS-CoV-2 nucleic acid detection kit

The sequences of the primers and the probes in the kit are shown in the following table 1: SEQ ID NO. 1-14 are primers and probes designed in the conserved regions of the N gene and ORF1ab gene of SARS-CoV-2.

Table 1: primer and probe sequence

The kit also comprises: PCR buffer, 10mM dNTPs, 200 u/. mu.l M-MLV enzyme, 5 u/. mu.l Taq enzyme, 50mM MgCl₂. The kit also includes a negative control (sterile water) and a positive control (artificially synthesized at a concentration of 1X 10)⁶Pseudovirus of Copies/ml).

Example 2 detection method of the kit of the present invention

The detection method is Real Time RT-PCR, the reaction process of the Real Time RT-PCR is (1) M-MLV enzyme reverse transcription, the Time is generally 10min, and the temperature is generally 50 ℃. (2) Pre-denaturation, wherein the time and the length depend on the length and the base composition of a target nucleic acid, the temperature of the pre-denaturation is generally 90-105 ℃, the time is generally 2-10 min, and the pre-denaturation aims to completely separate a double-stranded nucleic acid sequence into single strands; (3) denaturation, the temperature is generally 91-105 ℃, and the time is generally 5-35 s; (3) annealing, annealing each primer to the target sequence of the amplified nucleic acid. The annealing temperature is usually 40-60 ℃, the annealing time can be 10-60 s (4) for extension, the primer is combined with the template to begin synthesizing new DNA double strands, the extension temperature is usually 40-80 ℃, and the extension time can be 10 s-1 min.

The composition of each assay system is shown in table 2:

fluorescence detection channel selection: (1) selecting FAM channel (ReporTer: FAM, Quencher: none), detecting N gene of SARS-CoV-2; (2) ROX channel (ReporTer: ROX, Quencher: none) is selected, ORF1ab gene (3) for detecting SARS-CoV-2 and ReferenCe fluorescence (PAStive ReferenCe) are set as none.

The fluorescent quantitative real-time reaction conditions are shown in table 3 below.

Table 3: fluorescent quantitative real-time PCR reaction condition

The reaction time of the kit is 60-70 min, after the reaction is finished, the instrument automatically stores results, software carried by the instrument is used for carrying out automatic analysis or manual adjustment on a starting value, an ending value and a threshold value line value of a baseline for analysis, and then a sample CT value and a fixed value result are recorded. The specific test results were analyzed as follows:

EXAMPLE 3 feasibility test of the kit of the invention

1. Limit of detection (LOD) test

(1) SARS-CoV-2: SARS-CoV-2 nucleic acid detection reagent was prepared by the method of example 1.

(2) Pathogen sample extraction

200 mul of SARS-CoV-2 standard fire-fighting virus samples with different concentrations in the tube were taken out for nucleic acid extraction (nucleic acid extraction kit from Nanjing Riming biological products Co., Ltd.) to obtain nucleic acid templates for use.

(3) Sample detection

Mu.l of the treated sample nucleic acid was put into a SARS-CoV-2 nucleic acid detecting reagent reaction tube, and 10. mu.l of purified water was added to the detection solution as a negative control, and detection was carried out in accordance with the detection method in example 2.

(4) Analysis of results

The detection of the fire extinguishing virus samples of SARS-CoV-2 at each concentration by using the kit prepared in example 1 and the detection method of example 2 revealed that both the detection sensitivity (LOD) N gene and ORF1ab gene of the present detection method were 20copies/ml, as shown in tables 4 and 5.

Table 4: confirmation of minimum detection limit of N gene

Sample concentration (copies/ml)	Detecting the number of repetitions	Number of positive tests	Rate of positive detection
				200000copies/ml	21	21	100％
20000copies/ml	21	21	100％
				2000copies/ml	21	21	100％
200copies/ml	21	21	100％
				20copies/ml	21	21	14.2％
10copies/ml	21	3	0.0％
				0copies/ml	21	0	0.0％

TABLE 5 identification of the minimum detection Limit of ORF1ab Gene

Sample concentration (copies/ml)	Detecting the number of repetitions	Number of positive tests	Rate of positive detection
				200000copies/ml	21	21	100％
20000copies/ml	21	21	100％
				2000copies/ml	21	21	100％
200copies/ml	21	21	100％
				20copies/ml	21	20	95.5％
10copies/ml	21	2	9.5％
				0copies/ml	21	0	0.0％

EXAMPLE 4 comparison of the sensitivity of the kits of the invention with that of the comparative kit

(1) Pathogen sample extraction: samples of SARS-CoV-2 inactivated virus at 6 different concentrations in the tube were taken out by 200. mu.l each for nucleic acid extraction (nucleic acid extraction kit from Nanjing Riming Bioproducts Ltd.) to obtain nucleic acid templates for use.

(2) And sample detection, and parallel comparison detection of a comparison kit and the kit.

(2) The results are shown in FIGS. 1 to 4

Table 6: minimum detection limit comparison of N gene and ORF1ab gene of the invention and comparison kit

And (4) conclusion: compared with the PCR kit sold in the market, the primer probe provided by the invention has obvious detection sensitivity for the new coronavirus.

Example 5 detection specificity test of the kit of the invention

Other pathogens (coronavirus OC43, NL63, 229E, HKU1, parainfluenza virus, influenza A virus, adenovirus and respiratory syncytial virus) which are the same as the infection site of the novel coronavirus or have similar infection symptoms are detected, and the specificity of the amplification system is analyzed. Coronavirus OC43, NL63, 229E, HKU1, parainfluenza virus, influenza A virus, adenovirus and respiratory syncytial virus sample standard samples are purchased from Landao laboratory diagnosis Co., Ltd (Randox) in British, and the sample concentration is 10⁶-10⁷copies/mL。

The kit prepared by using example 1 and the detection method using example 2 were used. The detection result shows that no amplification curve exists for the eight viruses. The kit of the invention is proved to have no amplification to the eight viruses, and the specificity of the primer design of the invention is also verified.

Comparative example 1

Detection sensitivity test of other primer probes of the invention (SEQ ID NO.9 to 14)

The composition of each assay system is shown in table 2:

Table 3: fluorescent quantitative real-time PCR reaction condition

After the reaction is finished, the instrument automatically stores the result, automatically analyzes the initial value, the end value and the threshold value line value of the baseline by utilizing the software of the instrument or manually adjusts the initial value, the end value and the threshold value line value, and then records the CT value and the fixed value result of the sample. The specific test results were analyzed as follows:

Table 7: confirmation of minimum detection limits of N Gene and ORF1ab Gene in comparative example kit of the invention

And (4) conclusion: the primer probe provided by the invention has obvious detection sensitivity to the new coronavirus.

Sequence listing

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Claims

1. A primer and a probe for detecting SARS-CoV-2, which is characterized in that: the primer probe combination comprises the following components:

the upstream primer of SARS-CoV-2N gene shown in SEQ ID NO. 1;

the downstream primer of SARS-CoV-2N gene shown in SEQ ID NO. 2;

a specific probe of SARS-CoV-2N gene as shown in SEQ ID NO. 3;

the upstream primer of SARS-CoV-2ORF1ab gene shown in SEQ ID NO. 4;

the downstream primer of SARS-CoV-2ORF1ab gene shown in SEQ ID NO. 5;

and a specific probe of SARS-CoV-2ORF1ab gene as shown in SEQ ID NO. 6.

2. The primers and probe for detecting SARS-CoV-2 according to claim 1,

the segment amplified by the primer pair shown in SEQ ID NO. 1-2 is shown in SEQ ID NO. 7;

the fragments amplified by the primer pairs shown in SEQ ID NO. 4-5 are shown in SEQ ID NO. 8.

3. The primers and probe for detecting SARS-CoV-2 as claimed in claim 1, wherein the probe is linked to a quencher group at the 3 'end and a fluorophore group at the 5' end; wherein:

the 5' end of the probe shown as SEQ ID NO.3 is connected with FAM fluorophore;

4. Use of the primers and probes of any of claims 1 to 3 in the preparation of a kit for detecting SARS-CoV-2.

5. A SARS-CoV-2 detection kit, comprising the primer and probe of any one of claims 1 to 3 and Real time PCR reaction reagent.

6. The kit of claim 5, wherein the Real time PCR reaction reagents comprise: dNTPs, M-MLV enzyme, Taq enzyme, MgCl₂。

7. The kit of claim 5, further comprising a negative control and a positive control; wherein the negative control is sterile water, and the positive control is artificially synthesized with a concentration of 1 × 10⁶Pseudoviruses of Copies/ml.

8. A method for detecting SARS-CoV-2 for non-diagnostic or therapeutic purposes: detecting Real time PCR on a sample by using the primer and the probe of any one of claims 1 to 3, and judging whether the sample is infected by SARS-CoV-2 according to the Ct value detected;

the judgment comprises the following steps:

9. The method of claim 8,

the reaction system of Real time PCR comprises:

the reaction program of the Real time PCR comprises the following steps: