CN114134252B

CN114134252B - Primers and kit for detecting coronavirus

Info

Publication number: CN114134252B
Application number: CN202111214867.3A
Authority: CN
Inventors: 张舟; 林祥梅; 吴绍强; 吕继洲; 袁向芬; 王彩霞; 刘晓飞
Original assignee: Chinese Academy of Inspection and Quarantine CAIQ
Current assignee: Chinese Academy of Inspection and Quarantine CAIQ
Priority date: 2021-10-19
Filing date: 2021-10-19
Publication date: 2024-03-08
Anticipated expiration: 2041-10-19
Also published as: CN114134252A

Abstract

The invention provides a universal specific PCR detection primer pair for detecting coronaviruses and a specific PCR detection method for coronaviruses SADS-CoV viruses and SARS-CoV-2 viruses by using the primer pair. The primer pair for specifically detecting the coronavirus can specifically and simultaneously detect the SADS-CoV virus and the SARS-CoV-2 virus when the amplification is carried out by utilizing microdroplet digital RT-PCR, and can quickly and accurately absolute quantify the SADS-CoV or SARS-CoV-2 nucleic acid while greatly reducing the detection cost.

Description

Primers and kit for detecting coronavirus

Technical Field

The invention relates to the technical field of biology, in particular to a specific PCR detection primer pair for detecting coronaviruses, a method for detecting the coronaviruses by utilizing the detection primer pair in a microdroplet digital PCR mode and a kit.

Background

Coronaviruses belong to the genus Coronaviridae (Coronavirus) of the family Coronaviridae (Nidovirales) of the order Coronavirales in the phylogenetic classification. Is an RNA virus with envelope and linear single positive strand genome. Based on the genomic characteristics and genetic relationships of coronaviruses, the International Commission on viral classification (ICTV) classifies coronaviruses into the alpha, beta, gamma and delta 4 genera. Coronaviruses have a very broad host range of infections, can infect birds, humans and a wide variety of mammalian animals, and cause varying degrees of gastrointestinal and respiratory tract related diseases. Currently, 6 coronaviruses are known to infect pigs and 7 coronaviruses are known to infect humans.

SADS-CoV has genetic relationship with reported strain HKU-2 of bat coronavirus, and amino acid homology is more than 90%. SADS-CoV can be infected alone or in combination with porcine epidemic diarrhea virus (porcine epidemic diarrhea virus, PEDV), porcine delta coronavirus (porcine deltacoronavirus, PDCoV), porcine transmissible gastroenteritis virus (porcine transmissible gastroenteritis virus, TGEV) and Rotavirus (RV). At present, although there is no direct evidence that SADS-CoV can infect humans, but it can infect various cell lines such as bats, mice, rats, gerbils, hamsters, pigs, chickens, non-human primates, and humans, suggesting that it is potentially threatening for cross-species transmission. Up to the present, new coronavirus infection of pigs caused by SADS-CoV is popular in Guangdong, fujian and other places and in some foreign pig farms, and causes serious threat to pig industry and national gate biosafety guarantee work in China.

SARS-CoV-2 may be bats in origin, but it has been reported that many species are found to be infectable, and it has been demonstrated that tigers, minks, pangolins, snakes, ferrets, cats and dogs, etc. are infectable, and that, for example, ferrets, hamsters, macaques and some other mammals are susceptible to SARS-CoV-2 to the same extent as humans, and thus, infection with SARS-CoV-2 is not a biological obstacle for many species of mammals. The recent report in the journal of nature shows that more than one third of white deer in northeast of the united states has antibodies against SARS-CoV-2, indicating that they have been infected with a novel coronavirus, which is the first time that wild animals are found to be in widespread contact with the novel coronavirus, researchers have worried about the emergence of a new population of animals carrying SARS-CoV-2, once it emerges, viruses can evolve in the new population, and at the same time, more animals may become viral reservoirs, re-transmitting the virus to humans.

Currently, fluorescent quantitative RT-PCR is still a recognized "gold standard" for the definitive diagnosis of SADS-CoV and SARS-CoV-2 infection. For new epidemic diseases and external epidemic diseases, a large amount of manpower and material resources are consumed for monitoring the various epidemic diseases respectively, and epidemic disease prevention and control and port monitoring work are not facilitated in time, so that the orderly production in China and smooth implementation of national gate biosafety guarantee work are affected. The current widely used SARS-CoV-2 detection method is mostly a multi-target fluorescent RT-PCR detection method, and the purpose of detecting multiple targets of the same pathogen by adopting multiple RT-PCR is to increase detection specificity and reduce and prevent false positive, but the amplification efficiency and detection sensitivity are correspondingly reduced, and the detection cost is greatly increased. The single-target fluorescent RT-PCR has higher amplification efficiency and detection sensitivity, and lower detection cost, and can be used as a preferential selection scheme on the premise of ensuring the detection specificity.

Meanwhile, the quantitative method of fluorescent quantitative RT-PCR needs to rely on the setting of reference genes and standard samples, and can only achieve the aim of 'relative quantification', and cannot realize absolute quantification on low-concentration virus samples, and more importantly, cannot realize accurate detection in early stage of virus infection.

Therefore, there is no reagent capable of rapidly and accurately detecting SADS-CoV virus and SARS-CoV-2 virus.

Disclosure of Invention

In order to solve the above problems, the present invention aims to provide a reagent for coronaviruses, wherein the coronaviruses include SADS-CoV virus (porcine acute diarrhea syndrome coronavirus) and SARS-CoV-2 virus (severe acute respiratory syndrome coronavirus type 2), i.e., a reagent for detecting SADS-CoV virus and SARS-CoV-2 virus, comprising a primer combination specifically amplifying SADS-CoV virus and SARS-CoV-2 virus, the primer combination comprising a first primer pair and a second primer pair, the sequences of the first primer pair and the second primer pair being as follows:

a first primer pair:

SADS-CoV-F1：5’-CACTCATGGGCTGGGATT-3’，

SADS-CoV-R3：5’-GAACCGAGAACCATAGCAGC-3’；

a second primer pair:

SARS-CoV-2-F1：5’-ACCTTATGGGTTGGGATT-3’，

SARS-CoV-2-R3：5’-CGAGCAAGAACAAGTGAGGC-3’。

further, the reagent further comprises a probe, preferably the probe is a double quenching probe; more preferably, the sequence of the double quenching probe is as follows:

5’-/56-FAM/TCCTAAATG/ZEN/TGATAGAGCTATGCCT/3IABkFQ/-3’。

on the other hand, the application also provides a specific PCR detection method of coronavirus, which comprises the steps of carrying out PCR amplification on a sample to be detected by utilizing the primer combination, and if the target fragment is obtained by amplification, the sample to be detected is coronavirus; if the target fragment is not obtained by amplification, the sample to be detected is not coronavirus;

the coronaviruses include SADS-CoV virus and SARS-CoV-2 virus.

Further, the method comprises the step of performing amplification detection on the sample to be detected by using a microdroplet digital PCR system.

Preferably, the droplet digital PCR system described herein may employ existing commercial droplet digital PCR systems, and the steps of operating with such systems may also employ operations suitable for such systems.

Among them, digital droplet PCR (ddPCR) is a new technique for detecting and quantifying nucleic acids. The principle is that the reaction system is subjected to microdroplet treatment before the reaction to form tens of thousands to hundreds of thousands of water-in-oil liquefied microdroplets, each microdroplet contains one or a plurality of copies of target molecules (DNA or RNA templates), then the target molecules are respectively subjected to PCR amplification in each microdroplet, and after the amplification is finished, the fluorescent signals of the respective reaction microdroplets are subjected to statistical analysis by a microdroplet detector. ddPCR is independent of CT value, so that the amplification efficiency is not affected, and after the PCR amplification is finished, the copy number of the nucleic acid is calculated according to the detected positive droplet number and the Poisson distribution principle, and absolute quantification can be realized without comparing a standard sample with a standard curve. ddPCR is an excellent technique for quantitatively detecting trace nucleic acid molecules with good reproducibility, and compared with RT-PCR, the method has high detection flux, strong specificity and high sensitivity, and can realize absolute quantification in a real sense.

Further, the method further comprises the step of adding a probe to the reaction system;

preferably, the probe is a double quenching probe;

more preferably, the sequence of the double quenching probe is as follows:

5’-/56-FAM/TCCTAAATG/ZEN/TGATAGAGCTATGCCT/3IABkFQ/-3’。

wherein, the double quenching probe is a double quenching probe formed by adding a second quenching group between the 9 th base and the 10 th base of the designed probe and combining with a 5 '-end fluorescent group and a 3' -end quenching group (for example, the invention uses the fluorescent group FAM and the quenching group ZEN ^TM The Iowa Black FQ forms a double-quenching probe), the quenching effect superior to that of a single-quenching probe can be realized, the detection sensitivity is improved by using a lower background and a higher fluorescence signal, and the false positive is reduced.

Further, the amplification comprises a step of annealing at a temperature of 52-62 ℃, preferably 53-56 ℃, more preferably 53.9 ℃.

Further, the conditions for the amplification are specifically: reverse transcription is carried out for 60min at 45 ℃; enzyme activation at 95℃for 10min; denaturation at 95℃for 30s, annealing at 53.9℃and extension for 1min, 40 cycles total; inactivating enzyme at 98deg.C for 10min; constant temperature at 4 ℃.

Further, the amplified reaction system is as follows: supermix 5. Mu.L; reverse Transcriptase 2 μL;300mM DTT 1. Mu.L; 20. Mu.M upstream and downstream primers each 0.8. Mu.L, 20. Mu.M probe 0.25. Mu.L; 1 μl of RNA template; ddH2O was made up to 20. Mu.L.

In another aspect, the present application also provides a specific PCR detection kit for coronaviruses, including SADS-CoV virus and SARS-CoV-2 virus, comprising the above-described reagent.

In another aspect, the application also provides the application of the reagent in preparing a kit for detecting SADS-CoV virus and SARS-CoV-2 virus.

The technical scheme provided by the application has the following beneficial effects:

1. the primer pair for specifically detecting the coronavirus can specifically and simultaneously detect the SADS-CoV virus and the SARS-CoV-2 virus when the amplification is carried out by utilizing microdroplet digital RT-PCR, and can quickly and accurately absolute quantify the SADS-CoV or SARS-CoV-2 nucleic acid while greatly reducing the detection cost.

2. The PCR detection method for SADS-CoV virus and SARS-CoV-2 virus provided by the application adopts a microdroplet digital PCR system to process and amplify and detect the sample to be detected, and the method has high detection flux, strong specificity and high sensitivity; moreover, on the basis of amplification detection by utilizing microdroplet digital PCR, the detection method provided by the application also adopts a double-quenching probe, realizes a quenching effect superior to that of a single-quenching probe, improves detection sensitivity by using a lower background and a higher fluorescent signal, and reduces false positive; on the basis, the detection method provided by the application also optimizes a reaction system and amplification conditions for amplification, so that the amplification effect is optimal when the detection primer provided by the application is used for microdroplet digital PCR amplification detection.

3. The kit for detecting SADS-CoV virus and SARS-CoV-2 virus, which is provided by the application, contains the detection primer and the double-quenching probe, is simple to operate, and is particularly suitable for the detection method for amplifying by utilizing microdroplet digital PCR.

Drawings

The accompanying drawings, which are included to provide a further understanding of the application and are incorporated in and constitute a part of this application, illustrate embodiments of the application and together with the description serve to explain the application and do not constitute an undue limitation to the application. In the drawings:

FIG. 1 is a graph of amplification efficiency for different SADS-CoV primers using SADS-CoV positive samples as templates;

FIG. 2 is a graph showing amplification efficiencies of different SARS-CoV-2 primers using a SARS-CoV-2 nucleic acid standard substance as a template;

FIG. 3 is a graph showing amplification efficiency of different SADS-CoV and SARS-CoV-2 primer combinations using SADS-CoV positive samples and SARS-CoV-2 nucleic acid standard as templates;

FIG. 4 is an amplification plot of a single quenching probe using a SADS-CoV positive sample as a template;

FIG. 5 is an amplification plot of a double quenching probe using a SADS-CoV positive sample as a template;

FIG. 6 is an amplification plot of a single quenching probe using SARS-CoV-2 nucleic acid standard as a template;

FIG. 7 is an amplification plot of a double-quenching probe using SARS-CoV-2 nucleic acid standard as a template;

FIG. 8 is an amplification plot of the specificity experiment in example 3;

FIG. 9 is an amplification plot of SADS-CoV sensitivity experiments in example 4;

FIG. 10 is an amplification result of the SADS-CoV sensitivity test in example 4;

FIG. 11 is an amplification plot of SARS-CoV-2 sensitivity assay in example 4;

FIG. 12 shows amplification results of SARS-CoV-2 sensitivity assay in example 4.

Detailed Description

In order to more clearly illustrate the general concepts of the present application, the following detailed description is given by way of example. In the following description, numerous specific details are set forth in order to provide a more thorough understanding of the present application. However, it will be apparent to one skilled in the art that the present application may be practiced without one or more of these details. In other instances, some features well known in the art have not been described in order to avoid obscuring the present application.

The experimental methods used in the following examples are conventional methods unless otherwise specified.

Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

Example 1 design of primers and probes for detection of coronaviruses SADS-CoV and SARS-CoV-2

The embodiment designs a universal microdroplet digital PCR detection primer and a probe aiming at porcine acute diarrhea syndrome coronavirus SADS-CoV and severe acute respiratory syndrome coronavirus type 2 SARS-CoV-2 (namely novel coronavirus), and the specific method is as follows:

by comparing and analyzing all 15 genomic sequences of SADS-CoV isolates published in GenBank, 62 genomic sequences of SARS-CoV-2 isolates, and related viral genomic sequences closely related to other coronaviruses (e.g., nucleic acid sequence exclusive analysis of the Bat SARS-like corenavirus and SARS-CoV closely related to SARS-CoV-2), a region of ORF1ab that is free of secondary structure and relatively conserved is selected, pairs of primers and probes are designed, the primer length is typically about 20 bases, the probe length is about 25-30 bases, there is no complementary sequence between and within primers, the primers and probes are as follows:

an upstream primer:

SADS-CoV-F1：5’-CACTCATGGGCTGGGATT-3’（18nt），

SADS-CoV-F2：5’-CAAAGTTTTATGGTGGTTGGGAC-3（23nt），

SADS-CoV-F3：5’-ATGCTCAAGAATTTGATGAATGATG-3’（25nt），

SARS-CoV-2-F1：5’-ACCTTATGGGTTGGGATT-3’（18nt），

SARS-CoV-2-F2：5’-GCAAATTCTATGGTGGTTGGC-3’（21nt），

a downstream primer:

SADS-CoV-R1：5’-TAGCAGCAAGCATTCGTATCAT-3’（22nt），

SADS-CoV-R2：5’-GAGAACCATAGCAGCAAGC-3’（19nt），

SADS-CoV-R3：5’-GAACCGAGAACCATAGCAGC-3’（20nt），

SARS-CoV-2-R1：5’-GTGAGGCCATAATTCTAAGCAT-3’（22nt），

SARS-CoV-2-R2：5’- AAGAACAAGTGAGGCCATA-3’（19nt），

SARS-CoV-2-R3：5’-CGAGCAAGAACAAGTGAGGC-3’（20nt），

TaqMan probe SADS-CoV & SARS-CoV-2-P1:

5’-FAM-CTAAATGTGATAGAGCCATGCCTA-BHQ1-3’，

IDT double quenching probe SADS-CoV & SARS-CoV2-P2:

5’-/56-FAM/TCCTAAATG/ZEN/TGATAGAGCTATGCCT/3IABkFQ/-3’。

wherein, the SADS-CoV and the SARS-CoV-2 primer have one-to-one correspondence and are positioned at the same position of each genome.

Primer and probe optimization:

the results of the amplification of the target sequence by the primer and the probe are shown in fig. 1-3, wherein the probes used for primer optimization are the double-quenching probes, and the results are as follows:

the amplification efficiency of SADS-CoV primers was compared using SADS-CoV positive samples as templates, and the results are shown in FIG. 1. In FIG. 1, numbers 1 to 6 represent the amplification result curves of F1 to R3, F2 to R3, F1 to R2, F2 to R2, F1 to R1, F3 to R3, respectively, and the results of FIG. 1 indicate that the SADS-CoV primers F1 to R3, F2 to R3 have the highest amplification efficiency.

Similarly, the amplification efficiency of SARS-CoV-2 primer was compared using SARS-CoV-2 nucleic acid standard as a template, and the results are shown in FIG. 2. In FIG. 2, numbers 1 to 4 represent the amplification result curves of F1 to R3, F2 to R3, F1 to R1, F2 to R1, respectively, and the results of FIG. 2 indicate that F1 to R3, F2 to R3 are most efficient in amplification.

Then, the amplification efficiency of the combination of SADS-CoV and SARS-CoV-2 primers was compared using the SADS-CoV positive sample and the SARS-CoV-2 nucleic acid standard substance as templates, and the results are shown in FIG. 3. In FIG. 3, numbers 1-3 use SADS-CoV as a template and numbers 1'-3' use SARS-CoV-2 as a template; the numbers 1 and 1' are the primers SADS-CoV F1-R3 and SARS-CoV-2 F1-R3; the numbers 2 and 2' are primers SADS-CoV F2-R3 and SARS-CoV-2 F2-R3; the numbers 3 and 3' are the primers SADS-CoV F1-R3 and SARS-CoV F2-R3. The results of FIG. 3 show that the primer combinations of the primer numbers 2 and 2 'and the primer combinations of the primer numbers 3 and 3' have different degrees of reduction in amplification efficiency compared with the single amplification of the primers, and are suspected to be caused by the interaction among a plurality of groups of the primers; the SADS-CoV F1-R3 and SARS-CoV-2 F1-R3 primer combination has the highest amplification efficiency, and has no difference with the amplification efficiency when the primers SADS-CoV F1-R3 or SARS-CoV-2 F1-R3 are amplified independently.

In amplification assays using microdroplet digital PCR, optimization of reaction conditions:

setting 8 temperature gradients of 62.0 ℃, 61.2 ℃, 60.0 ℃, 58.1 ℃, 55.8 ℃,53.9 ℃, 52.7 ℃ and 52.0 ℃ by taking SADS-CoV positive samples or SARS-CoV-2 nucleic acid standard substances as templates, and parallelly controlling FAM-BHQ1 single quenching probe and FAM-ZEN ^TM The amplification condition of the Iowa Black and FQ double-quenching probe optimizes the universal droplet digital RT-PCR detection method.

Wherein, SADS-CoV positive sample or SARS-CoV-2 nucleic acid standard substance is used as template, and the amplification condition of single quenching probe or double quenching probe is compared, the obtained result is shown in figures 4-7, specifically:

FIG. 7 is an amplification plot of a double-quenched probe using SARS-CoV-2 nucleic acid standard as a template.

Comparing the amplification results of FIGS. 4-7, it can be seen that under the same reaction conditions, the double-quenching probe has a lower amplification background value and a higher fluorescence signal, so that the detection sensitivity can be improved and the false positive can be reduced.

The reaction channels A03-H03 or A06-H06 of each of FIGS. 4-7 are in one-to-one correspondence with the 8 annealing temperature gradients, namely 62.0 ℃, 61.2 ℃, 60.0 ℃, 58.1 ℃, 55.8 ℃,53.9 ℃, 52.7 ℃ and 52.0 ℃ respectively. The interpretation method of the digital PCR result is as follows: the ordinate represents fluorescence intensity (mainly used for distinguishing negative from positive), and the width of the abscissa reflects the number of points in the graph, namely the number of positive droplets in the reaction system. Under the same conditions, the more positive droplets represents the higher efficiency of the amplification reaction. From the results of FIGS. 4 to 7, it was also found that the amplification was best when the annealing temperature in the reaction conditions was 53.9 ℃.

The optimal primer and probe sequence combinations were analyzed by control experiments as follows:

an upstream primer:

SADS-CoV-F1：5’-CACTCATGGGCTGGGATT-3’，

SARS-CoV-2-F1：5’-ACCTTATGGGTTGGGATT-3’，

a downstream primer:

SADS-CoV-R3：5’-GAACCGAGAACCATAGCAGC-3’，

SARS-CoV-2-R3：5’-CGAGCAAGAACAAGTGAGGC-3’，

IDT double quenching probe SADS-CoV & SARS-CoV2-P2:

5’-/56-FAM/TCCTAAATG/ZEN/TGATAGAGCTATGCCT/3IABkFQ/-3’。

example 2 PCR detection method for specific detection of coronaviruses SADS-CoV and SARS-CoV-2

This example provides the optimal primer and probe sequence combinations and optimized reaction conditions of example 1 for the specific detection of coronaviruses SADS-CoV and SARS-CoV-2 by a PCR detection method comprising the steps of:

step 1, extraction of total RNA in pig tissue or infected cells: extracting total RNA of a sample to be detected by using a QIAGEN RNeasy cube Plus Mini Kit (Cat No.: 74134);

extraction of total RNA from porcine serum, viral cell culture supernatant or human pharyngeal swab samples: total RNA of the sample to be tested was extracted using QIAGEN QIAamp Viral RNA Mini Kit kit (Cat No.: 52904).

Step 2, microdroplet digital RT-PCR amplification

The method uses a PCR amplification apparatus (Bio-Rad Co.) and a QX200 ™ Droplet Digital ™ PCR Systems (Bio-Rad Co.) and comprises the following amplification steps:

1) The optimized microdroplet digital RT-PCR amplification reactions were performed using the Bio-Rad One-Step RT-ddPCR ™ Advanced Kit for Probes (SDS) kit (Cat No.: 1864021): supermix 5. Mu.L; reverse Transcriptase 2 μL;300mM DTT 1. Mu.L; 20. Mu.M upstream and downstream primers each 0.8. Mu.L, 20. Mu.M probe 0.25. Mu.L; 1 μl of RNA template; ddH2O was made up to 20. Mu.L.

2) 20 mu L of the reaction system in the step 1) is added into 8 holes of a middle row of a droplet generation card (DG 8 cartridge) of the droplet digital RT-PCR, 70 mu L of droplet generation oil (DG oil) is added into the 8 holes of the lowest row of the DG8cartridge, a rubber cushion is covered, and the droplet generation is carried out in a droplet generator. The reaction system in micro-droplet form is transferred to 96-well plate to seal film.

3) Transferring the sealed 96-well plate into a PCR amplification instrument for amplification under the following conditions: reverse transcription is carried out for 60min at 45 ℃; enzyme activation at 95℃for 10min; denaturation at 95℃for 30sec, annealing at 53.9℃and extension for 1min, 40 cycles total; inactivating enzyme at 98deg.C for 10min; constant temperature at 4 ℃.

After the PCR amplification is finished, transferring the 96-well plate with the sealing film into a micro-droplet fluorescence detector for reading, calculating the quantity of micro-droplets emitting fluorescence in each tube of reaction system, and accurately calculating the copy numbers of SADS-CoV and SARS-CoV-2 in the sample according to the Poisson distribution principle.

Step 3, result judgment

The reading of the microdroplet fluorescence detector is not 0, namely the amplification signal in the microdroplet reaction system is detected, and the reaction well is considered to be a positive amplification well.

When the reaction hole of the positive quality control product generates a positive result and the reaction hole of the negative quality control product generates a negative result, the experiment is considered to be an effective experiment, otherwise, the experiment is ineffective, and the reaction reagent needs to be replaced or the reaction condition needs to be changed for carrying out again.

The criteria for positive samples were: at least one positive amplification well appears in 3 repeated reaction groups of the same sample, which indicates that the sample to be detected contains porcine acute diarrhea syndrome coronavirus or novel coronavirus.

The criteria for negative samples were: negative results in 3 repeated reaction groups of the same sample indicate that the sample to be detected does not contain porcine acute diarrhea syndrome coronavirus or novel coronavirus nucleic acid.

EXAMPLE 3 specificity experiments for microdroplet digital RT-PCR

In this example, the specificity of the primers and probes to SADS-CoV and SARS-CoV-2 was verified by using the micro-droplet digital RT-PCR method using the RNA and negative controls of porcine acute diarrhea syndrome coronavirus SADS-CoV, novel coronavirus SARS-CoV-2, porcine epidemic diarrhea virus PEDV, porcine transmissible gastroenteritis virus TGEV, porcine delta coronavirus PDCoV, porcine vesicular disease virus SVDV, foot and mouth disease virus FMDV, A-type Seika virus SVA, porcine reproductive and respiratory syndrome PRRSV, porcine pestivirus CSFV, porcine sapelo virus PSV as templates, and the results obtained are shown in FIG. 8, and in FIG. 8, the results of the B01-B12 reaction channels correspond to the above viruses one by one, respectively.

The results in FIG. 8 show that SADS-CoV and SARS-CoV-2 samples are amplified by microdroplet digital RT-PCR, typical amplification signals appear, and no specific amplification signals appear in the rest samples, thus indicating that the microdroplet digital RT-PCR primers and probes of the invention have good specificity.

Example 4 sensitivity experiment of microdroplet digital RT-PCR

In this example, a 10-fold gradient dilution of SADS-CoV positive samples and SARS-CoV-2 nucleic acid standard (cRNA, standard substance number: GBW (E) 091089, available from China national institute of metrology) was used as a template, and detection was performed using microdroplet digital RT-PCR, and the results are shown in FIGS. 9 to 12.

The results of FIGS. 9 and 10 show that the detection limit is 1.2 copies/. Mu.L using the SADS-CoV sample as a template.

The results in FIGS. 11 and 12 show that the detection limit was 1.4 copies/. Mu.L using SARS-CoV-2 nucleic acid standard as a template.

The results of FIGS. 9-12 demonstrate that the microdroplet digital RT-PCR primers and probes provided after the screening of example 1 are highly sensitive.

Example 5 repeat experiments of microdroplet digital RT-PCR

In this example, a total of 6 gradients of SADS-CoV positive samples and SARS-CoV-2 nucleic acid standard substances 1.0E+05, 1.0E+04, 1.0E+03, 1.0E+02, 1.0E+01, and 1.0E+00 copies/. Mu.L were used as templates, and a droplet digital RT-PCR method was used to perform intra-and inter-group reproducibility experiments, each experiment was performed in 3 replicates for a total of 3 experiments. The results obtained are shown in tables 1 and 2.

TABLE 1 SADS-CoV repeatability experiments

TABLE 2 SARS-CoV-2 repeatability experiments

The results in tables 1 and 2 show that the microdroplet digital RT-PCR primers and probes of the invention have good reproducibility.

Therefore, when the detection primer provided by the application is used for amplification by using the optimized microdroplet digital RT-PCR provided by the application, the SADS-CoV virus and the SARS-CoV-2 virus can be detected simultaneously and specifically, the specificity is good, the sensitivity is high, the repeatability is good, and the SADS-CoV or SARS-CoV-2 nucleic acid can be rapidly and accurately absolute quantified.

The foregoing is merely exemplary of the present application and is not intended to limit the present application. Various modifications and changes may be made to the present application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc. which are within the spirit and principles of the present application are intended to be included within the scope of the claims of the present application.

SEQUENCE LISTING

<110> national institute of inspection and quarantine science

<120> primers and kit for detection of coronavirus

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<170> PatentIn version 3.5

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Claims

1. A reagent for detecting SADS-CoV virus and SARS-CoV-2 virus, comprising a primer combination for specifically amplifying SADS-CoV virus and SARS-CoV-2 virus,

the primer combination comprises a first primer pair and a second primer pair, and the sequences of the first primer pair and the second primer pair are as follows:

a first primer pair:

SADS-CoV-F1：5’-CACTCATGGGCTGGGATT-3’，

SADS-CoV-R3：5’-GAACCGAGAACCATAGCAGC-3’；

a second primer pair:

SARS-CoV-2-F1：5’-ACCTTATGGGTTGGGATT-3’，

SARS-CoV-2-R3：5’-CGAGCAAGAACAAGTGAGGC-3’；

the reagent further comprises a double quenching probe, and the sequence of the double quenching probe is as follows:

5’-/56-FAM/TCCTAAATG/ZEN/TGATAGAGCTATGCCT/3IABkFQ/-3’。

2. a universal specific PCR detection kit for coronaviruses, said coronaviruses being SADS-CoV virus and SARS-CoV-2 virus, characterized in that said kit comprises the reagent of claim 1.

3. Use of the reagent of claim 1 in the preparation of a kit for detecting SADS-CoV virus and SARS-CoV-2 virus.