Patent Application
20230193407
The contents of the electronic sequence listing (CU693 SequenceListing.xml; Size: 65,593 bytes; and Date of Creation: Aug. 19, 2022) is herein incorporated by reference in its entirety.
The present invention relates to the field of molecular biological detection, more specifically, to the field of detection of coronaviruses. More specifically, the present invention is capable of detecting and typing novel coronavirus 2019-nCoV, coronavirus 229E, coronavirus NL63, coronavirus OC43, coronavirus HKU1, coronavirus MERSr-CoV, and coronavirus SARSr-CoV.
Coronaviruses are nonsegmented, single stranded and positive-stranded RNA viruses, which belong to the Orthocoronavirinae subfamily of the Coronaviridae family of the order Nidovirales. According to serotypes and genome characteristics, the coronavirus subfamily is divided into four genera: α, β, γ, and δ, and can infect many animal species, including humans, bats, dogs, pigs, mice, birds, cattle, whales, horses, goats, monkeys, etc. There are six coronaviruses known to infect humans, including 229E and NL63 of the genus α, OC43 and HKU1 of the genus β, the Middle East respiratory syndrome-related coronavirus (MERSr-CoV), and the severe acute respiratory syndrome-related coronavirus (SARSr-CoV). In December 2019, an unexplained pneumonia infection event occurred in Wuhan, Hubei, and a new type of coronavirus belonging to the genus (3 was isolated from the lower respiratory tract of patients. Subsequently, the World Health Organization named the virus “2019 Novel Coronavirus (2019-nCoV).”
The novel coronavirus is a novel coronavirus of the genus (3, which has an envelope and round or oval particles, often pleomorphic, with a diameter of 60-140 nm. The genetic characteristics thereof are significantly different from those of SARSr-CoV and MERSr-CoV. According to the latest research results of researcher Shi Zhengli from the Wuhan Institute of Virology, 2019-nCoV is highly similar to RaTG13, a bat coronavirus strain previously isolated from Chinese rufous horseshoe bats in her laboratory, with an overall genomic similarity of 96.2%. In addition, studies have shown that 2019-nCoV is 85% or more homologous to bat SARS-like coronaviruses (bat-SL-CoVZC45 and bat-SL-CoVZXC21).
Among the seven coronaviruses that can infect humans, coronavirus 229E, coronavirus NL63, coronavirus OC43, and coronavirus HKU1 are common respiratory infectious viruses, which cause mild cases after infecting people and usually do not induce severe illnesses. However, novel coronavirus 2019-nCoV, coronavirus MERSr-CoV, and coronavirus SARSr-CoV infections will cause very serious infection symptoms in patients, and are highly likely to develop into severe illnesses. If various coronaviruses can be clearly typed in a single test, subsequent diagnosis and treatment measures will be greatly facilitated, to achieve precise treatment, prevention and control, especially for the highly contagious and harmful novel coronavirus 2019-nCoV, coronavirus MERSr-CoV, and coronavirus SARSr-CoV, making it possible to control the source of infection in a timely manner and block virus pandemics and outbreaks.
Therefore, there is a need in the art for a related product that can perform simultaneous and rapid identification and typing of multiple coronaviruses.
In view of this, provided in the present invention is a composition capable of detecting and typing coronaviruses, the composition including:
a first group:
a novel coronavirus 2019-nCoV forward primer as shown in SEQ ID NO: 1, a novel coronavirus 2019-nCoV reverse primer as shown in SEQ ID NO: 2, and a novel coronavirus 2019-nCoV probe as shown in SEQ ID NO: 3;
a coronavirus NL63 forward primer as shown in SEQ ID NO: 4, a coronavirus NL63 reverse primer as shown in SEQ ID NO: 5, and a coronavirus NL63 probe as shown in SEQ ID NO: 6;
a coronavirus HKU1 forward primer as shown in SEQ ID NO: 7, a coronavirus HKU1 reverse primer as shown in SEQ ID NO: 8, and a coronavirus HKU1 probe as shown in SEQ ID NO: 9; and
a second group:
a coronavirus 229E forward primer as shown in SEQ ID NO: 10 and a coronavirus 229E reverse primer as shown in SEQ ID NO: 11;
a coronavirus OC43 forward primer as shown in SEQ ID NO: 12 and a coronavirus OC43 reverse primer as shown in SEQ ID NO: 13;
a coronavirus MERSr-CoV forward primer as shown in SEQ ID NO: 14 and a coronavirus MERSr-CoV reverse primer as shown in SEQ ID NO: 15; and
a coronavirus SARSr-CoV forward primer as shown in SEQ ID NO: 16 and a coronavirus SARSr-CoV reverse primer as shown in SEQ ID NO: 17,
wherein fluorescent groups in the first group are different from one another, and fluorescent groups in the second group are different from one another.
The coronaviruses includes: novel coronavirus 2019-nCoV, coronavirus 229E, coronavirus NL63, coronavirus OC43, coronavirus HKU1, coronavirus MERSr-CoV, and coronavirus SARSr-CoV.
Using the composition of the present invention in combination with a fluorescent probe method and a melting curve method, it is possible to use one tube to perform simultaneous detection and typing of seven coronaviruses in one test, achieving low costs, high throughput, and less time consumption. The present invention enables information of eight targets to be given by one tube in a single test, greatly improving the detection efficiency, and the operations are simple and convenient, and a result reading process can be determinated according to an amplification curve and a CT value. The whole detection process is carried out under single-tube closed conditions, avoiding false positives and environmental contamination caused by crossover between samples.
In the present invention, the fluorescent reporter group may be selected from FAM, HEX, ROX, VIC, CY5, 5-TAMRA, TET, CY3, and JOE, but is not limited thereto.
In one specific embodiment, the fluorescent reporter group of the novel coronavirus 2019-nCoV probe as shown in SEQ ID NO: 3 is FAM; the fluorescent reporter group of the coronavirus NL63 probe as shown in SEQ ID NO: 6 is HEX; and the fluorescent reporter group of the coronavirus HKU1 probe as shown in SEQ ID NO: 9 is ROX.
In one specific embodiment, the fluorescent reporter group of the coronavirus 229E forward primer as shown in SEQ ID NO: 10 is FAM; the fluorescent reporter group of the coronavirus OC43 forward primer as shown in SEQ ID NO: 12 is HEX; the fluorescent reporter group of the coronavirus MERSr-CoV forward primer as shown in SEQ ID NO: 14 is ROX; and the fluorescent reporter group for the coronavirus SARSr-CoV as shown in SEQ ID NO: 16 is CY5.
In one embodiment, the components of the composition of the present invention are in the same package.
Further, the composition further includes: an internal standard forward primer as shown in SEQ ID NO: 18, an internal standard reverse primer as shown in SEQ ID NO: 19, and an internal standard probe as shown in SEQ ID NO: 20.
In one specific embodiment, a fluorescent reporter group of the internal standard probe as shown in SEQ ID NO: 20 is CY5.
Further, the amount of the primer in the composition is 50-150 nM, and the amount of the probe in the composition is 25-75 nM.
In a second aspect, provided in the present invention is a use of the composition of the present invention in the preparation of a kit for detecting and typing coronaviruses.
The coronavirus includes: novel coronavirus 2019-nCoV, coronavirus 229E, coronavirus NL63, coronavirus OC43, coronavirus HKU1, coronavirus MERSr-CoV, and coronavirus SARSr-CoV.
In a third aspect, provided in the present invention is a kit for detecting and typing coronaviruses, the kit including the composition of the present invention.
Further, the kit further includes at least one of a nucleic acid release reagent, a dNTP, a reverse transcriptase, a DNA polymerase, a PCR buffer, and Mg2+.
Further, the amount of the primer in the composition is 50-150 nM, the amount of the probe in the composition is 25-75 nM, and the amount of the dNTP is 0.2-0.3 mM.
Further, the concentration of the reverse transcriptase is 5 U/μL to 15 U/μL, and for example, the reverse transcriptase may be a murine leukemia reverse transcriptase (MMLV). The concentration of the DNA polymerase is 5 U/μL to 15 U/μL, and for example, the DNA polymerase may be a Taq enzyme.
In one specific embodiment, in addition to the above composition of the present invention, the kit further includes the following components and amounts:
In a fourth aspect, a method for detecting and typing coronaviruses is provided, the method including the steps of:
1) releasing a nucleic acid of a testing sample;
2) performing, by using the above composition of the present invention or the above kit of the present invention, a fluorescent quantitative PCR on the nucleic acids obtained in step 1); and
3) obtaining and analyzing the results.
In the present invention, the sample for detection may be a throat swab, sputum, a bronchoalveolar lavage fluid, blood, etc., but is not limited thereto.
Further, reaction conditions of the fluorescent quantitative PCR are as follows:
In a variant of the present invention, optionally, the novel coronavirus 2019-nCoV forward primer may be as shown in SEQ ID NO: 24, the novel coronavirus 2019-nCoV reverse primer may be as shown in SEQ ID NO: 25, and the novel coronavirus 2019-nCoV probe may be shown as
SEQ ID NO: 26; the coronavirus NL63 forward primer may be as shown in SEQ ID NO: 30, the coronavirus NL63 reverse primer may be as shown in SEQ ID NO: 31, and the coronavirus NL63 probe may be as shown in SEQ ID NO: 32; the coronavirus HKU1 forward primer may be as shown in SEQ ID NO: 36, the coronavirus HKU1 reverse primer may be as shown in SEQ ID NO: 37, and the coronavirus HKU1 probe may be as shown in SEQ ID NO: 38; the coronavirus 229E forward primer may be as shown in SEQ ID NO: 39, and the coronavirus 229E reverse primer may be as shown in SEQ ID NO: 40; the coronavirus OC43 forward primer may be as shown in SEQ ID NO: 45, and the coronavirus OC43 reverse primer may be as shown in SEQ ID NO: 46; the coronavirus MERSr-CoV forward primer may be as shown in SEQ ID NO: 49, and the coronavirus MERSr-CoV reverse primer may be as shown in SEQ ID NO: 50; and/or the coronavirus SARSr-CoV forward primer may be as shown in SEQ ID NO: 51, and the SARSr-CoV reverse primer may be as shown in SEQ ID NO: 52.
The present invention will be described in detail below with reference to specific embodiments and examples, and the advantages and various effects of the present invention will be more clearly presented therefrom. It should be understood by those skilled in the art that these specific embodiments and examples are intended to illustrate the present invention, rather than to limit the present invention.
The primers and probes used in the present invention are shown in Tables 1-3 below:
Among them, a fluorescent reporter group of the novel coronavirus 2019-nCoV probe was FAM; a fluorescent reporter group of the coronavirus NL63 probe was HEX; a fluorescent reporter group of the coronavirus HKU1 probe was ROX, and the 3′-end of the probes further had a BHQ1 or BHQ2 quencher group.
A fluorescent reporter group of the coronavirus 229E forward primer was FAM; a fluorescent reporter group of the coronavirus OC43 forward primer was HEX; a fluorescent reporter group of the coronavirus MERSr-CoV forward primer was ROX; and a fluorescent reporter group of the coronavirus SARSr-CoV was CY5.
A testing sample of the present invention was a throat swab, sputum, a bronchoalveolar lavage fluid, or blood. A magnetic bead method was used to extract a viral nucleic acid, and the following operations were performed in a sample processing room:
2.1 An appropriate number of 1.5-mL sterilized centrifuge tubes were taken, and labeled as a negative control, a positive control, and a testing sample, respectively. 300 μL of an RNA extraction solution 1 was added to each tube.
2.2 200 μL of the testing sample or the negative control or the positive control was added to each tube. The tube was covered with a cap, and shaken for 10 seconds for through mixing, and subjected to instant centrifugation.
2.3 100 μL of an RNA extraction solution 2-mix was added to each tube (sucked up after sufficient mixing), and the tube was shaken for 10 seconds for through mixing, and left to stand for 10 minutes at room temperature.
2.4 After instant centrifugation, the centrifuge tubes were placed on a separator, and the solution was slowly sucked out after 3 minutes (be careful not to touch a brown substance adhered to the tube wall).
2.5 600 μL of an RNA extraction solution 3 and 200 μL of an RNA extraction solution 4 were added to each tube, and the tube was shaken for 5 seconds for through mixing and subjected to instant centrifugation, and then the centrifuge tube was placed on the separator again.
2.6 After about 3 minutes, the supernatant separated into two layers. A pipette tip was inserted into the bottom of the centrifuge tube, the liquid was slowly sucked up from the bottom completely and discarded. The tube was left to stand for 1 minute and then the residual liquid at the bottom of the tube was completely sucked up and discarded.
2.7 50 μL of PCR-mix was added to each tube, and a pipette tip was used to suck up the PCR-mix to elute the brown residue adhered to the wall of the centrifuge tube. The operation was repeated several times to elute the residue as completely as possible, and then all the eluted brown mixture was transferred to a 0.2-mL PCR reaction tube, and the tube was covered with a cap and transferred to an amplification detecting zone.
The real-time fluorescent PCR reaction system was configured as follows:
The PCR amplification program was set up as follows:
Result analysis:
1) Target detection signals were FAM, HEX (or VIC), ROX, and CY5, and an internal reference detection signal was CY5.
2)Baseline setting: The baseline was generally set to 3-15 cycles, which specifically may be adjusted according to actual situations. The adjustment principle was to select a region where a fluorescence signal is relatively stable before exponential amplification, a starting point (Start) avoiding signal fluctuation in an initial stage of fluorescence collection, and an ending point (End) being less by 1-2 cycles than the Ct of a sample showing earliest exponential amplification. Threshold setting: the setting principle was to make a threshold line just exceed the highest point of a normal negative control.
3) It was first analyzed whether an amplification curve for the internal standard is detected in the CY5 channel and Ct≤39, and if so, it indicated that the current test was effective, and subsequent analysis continued to be carried out:
A) if a typical S-shaped amplification curve was detected in the FAM channel and Ct<39, it indicated that the novel coronavirus 2019-nCoV detection result was positive; if a characteristic peak of Tm (69.5±1.0° C.) was detected in the FAM channel, it indicated that the coronavirus 229E detection result was positive;
B) if a typical S-shaped amplification curve was detected in the HEX channel and Ct<40, it indicated that the coronavirus NL63 detection result was positive; if a characteristic peak of Tm (67.0±1.0° C.) was detected in the HEX channel, it indicated that the coronavirus OC43 was positive;
C) if a typical S-shaped amplification curve was detected in the ROX channel and Ct<40, it indicated that the coronavirus HKU1 detection result was positive; if a characteristic peak of Tm (70.5±1.0° C.) was detected in the ROX channel, it indicated that the coronavirus MERSr-CoV detection result was positive; and
D) if a characteristic peak of Tm (68.0±1.0° C.) was detected in the CY5 channel, it indicated that the coronavirus SARSr-CoV detection result was positive.
4) If a Ct for the internal standard was not detected in the CY5 channel or Ct>39, it indicated that the concentration of the testing sample was excessively low or there was an interfering substance that inhibited the reaction, and the experiment needed to be re-prepared.
Embodiment 3. Detection Results of Detecting the Positive Control by the Composition of the Present Invention
The compositions in Tables 1 to 3 of the present invention were used to test each target-positive plasmid according to the method described in Example 2, so as to simulate a clinical sample. Multiple PCR tests were conducted on a Hongshi fluorescent quantitative PCR instrument. The results are as shown in
When the coronavirus 2019-nCoV was detected with the composition in Table 1, the Ct value was around 25; when the coronavirus 2019-nCoV was detected with the composition in Table 2, the Ct value was around 28; and when the coronavirus 2019-nCoV was detected with the composition in Table 3, the Ct value was around 32. The amplification curves of A, B, and C were all relatively steep.
When the coronavirus NL63 was detected with the composition in Table 1, the Ct value was around 30; when the coronavirus NL63 was detected with the composition in Table 2, the Ct value was around 29, but the curve of B was not as steep as that of A; and when the coronavirus NL63 was detected with the composition in Table 3, the Ct value was around 34, and there was basically no amplification curve.
When the coronavirus HKU1 was detected with the composition in Table 1, the Ct value was around 25; when the coronavirus HKU1 was detected with the composition in Table 2, the Ct value was around 30; and when the coronavirus HKU1 was detected with the composition in Table 3, the Ct value was around 34, and the amplification curves of A and B were both relatively steep, and C basically had no amplification curve.
When the coronavirus 229E was detected with the composition in Table 1, the characteristic peak was obvious; when the coronavirus 229E was detected with the composition in Table 2, the characteristic peak was relatively obvious; and when the coronavirus 229E was detected with the composition in Table 3, the characteristic peak was not obvious.
When the coronavirus OC43 was detected with the composition in Table 1, the characteristic peak was obvious; when the coronavirus OC43 was detected with the composition in Table 2, the characteristic peak was relatively obvious; and when the coronavirus OC43 was detected with the composition in Table 3, the characteristic peak was not obvious.
When the coronavirus MERSr-CoV was detected with the composition in Table 1, the characteristic peak was obvious; when the coronavirus MERSr-CoV was detected with the composition in Table 2, the characteristic peak was relatively obvious; and when the coronavirus MERSr-CoV was detected with the composition in Table 3, the characteristic peak was not obvious.
When the coronavirus SARSr-CoV was detected with the composition in Table 1, the characteristic peak was obvious; when the coronavirus SARSr-CoV was detected with the composition in Table 2, the characteristic peak was relatively obvious; and when the coronavirus SARSr-CoV was detected with the composition in Table 3, the characteristic peak was not obvious.
When the internal standard was detected with the composition in Table 1, the Ct value was around 27; when the internal standard was detected with the composition in Table 2, the Ct value was around 30; and when the internal standard was detected with the composition in Table 3, the Ct value was around 31, and the amplification curves of A and B were both relatively steep, while the amplification curve of C was not steep.
| Number | Date | Country | Kind |
|---|---|---|---|
| 202010136909.5 | Mar 2020 | CN | national |
This application is a continuation of International Application No. PCT/CN2020/090054, filed on May 13, 2020, which claims priority to Chinese Patent Application No. 202010136909.5, filed on Mar. 2, 2020. The disclosures of the aforementioned applications are hereby incorporated by reference in their entireties.
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/CN2020/090054 | May 2020 | US |
| Child | 17891291 | US |
