COMPOSITION, KIT FOR DETECTING MUTATIONS OF 2019 NOVELCORONAVIRUS AND USE THEREOF
20230220452 · 2023-07-13
Inventors
- Lizhong DAI (Changsha, CN)
- Zhongping DENG (Changsha, CN)
- Jin YAN (Changsha, CN)
- Shiyao CHEN (Changsha, CN)
- Deyong TAN (Changsha, CN)
- Jia LIU (Changsha, CN)
- Xiaomei REN (Changsha, CN)
- Xinwu GUO (Changsha, CN)
Cpc classification
International classification
Abstract
Provided is a composition for detecting mutations of 2019 novel coronavirus mutation. Also provided are a kit containing the composition, the use of the composition, and a method for detecting mutations of 2019 novel coronavirus.
Claims
1. A composition for detecting mutations of 2019 novel coronavirus, comprising: a first nucleic acid composition: TABLE-US-00013 8782 C-type forward primer: (SEQ ID NO: 1) 5′-GCTGATTTTGACACATGGTTCATC-3′; and 28144 T-type forward primer: (SEQ ID NO: 2) 5′-ATCGGTAATTATACAGTTTCCAGATT-3′; and a second nucleic acid composition: TABLE-US-00014 8782 T-type forward primer: (SEQ ID NO: 3) 5′-GCTGATTTTGACACATGGTATCGT-3′; and 28144 C-type forward primer: (SEQ ID NO: 4) 5′-ATCGGTAATTATACAGTTTCCTTTGC-3′; wherein each nucleic acid composition further comprises: TABLE-US-00015 8782 reverse primer: (SEQ ID NO: 5) 5′-CCATTAGTTGTGCGTAATATCGT-3′; 8782 probe: (SEQ ID NO: 6) 5′-CTTGCCCATTGATTGCTGCAGTCATAA-3′; 28144 reverse primer: (SEQ ID NO: 7) 5′-CAACACGAACGTCATGATACTCTA-3′; and 28144 probe: (SEQ ID NO: 8) 5′-ATTGCCAGGAACCTAAATTGGGTAGT-3′.
2. The composition according to claim 1, wherein the 8782 probe and the 28144 probe carry fluorescent reporter groups that do not interfere with each other.
3. The composition according to claim 2, wherein the fluorescent reporter group is selected from a group consisting of FAM, HEX, ROX, VIC, CY5, 5-TAMRA, TET, CY3, and JOE.
4. The composition according to claim 1, wherein the fluorescent reporter group of the 8782 probe is HEX; and the fluorescent reporter group of the 28144 probe is FAM.
5. The composition according to claim 1, wherein a dosage of the primer in the composition is 25 nM-800 nM.
6. The composition according to claim 1, wherein a dosage of the probe in the composition is 10 nM-500 nM.
7. The composition according to claim 1, wherein the first nucleic acid composition and the second nucleic acid composition are in different packages.
8. The composition according to claim 1, wherein the two nucleic acid compositions combined are respectively present in a separate package.
9. The composition according to claim 1, further comprising an internal standard upstream primer, an internal standard downstream primer, and an internal standard probe for monitoring.
10. The composition according to claim 1, wherein a lowest detection concentration of the two nucleic acid composition is 1.0E+03 copy/mL.
11. A kit for detecting mutations of 2019 novel coronavirus, comprising the composition according to claim 1.
12. The kit according to claim 11, comprising a positive control.
13. The kit according to claim 11, wherein the positive control comprises sequences near an 8782-nd site and sequences near a 28144-th site of a 2019 novel coronavirus genome, and wherein the 8782-nd site is C or T, and the 28144-th site is C or T.
14. The kit according to claim 12, wherein the positive control further comprises an insertion sequence SEQ ID NO: 9.
15. The kit according to claim 12, wherein the positive control further comprises an insertion sequence SEQ ID NO: 10.
16. The kit according to claim 11, wherein a dosage of the primer in the composition is 25 nM-800 nM.
17. The kit according to claim 11, wherein a dosage of the probe in the composition is 10 nM-500 nM.
18. A method for detecting mutations of 2019 novel coronavirus, comprising the following steps: 1) releasing nucleic acids from a to-be-tested sample; 2) using the composition of claim 1 to perform fluorescent quantitative PCR on the nucleic acids obtained in step 1); and 3) obtaining and analyzing a result.
19. The method according to claim 18, wherein the sample is selected from a group consisting of a throat swab, sputum, bronchoalveolar lavage fluid and blood.
20. The method according to claim 18, wherein a dosage of the primer in the composition is 25 nM-800 nM, and a dosage of the probe in the composition is 10 nM-500 nM.
Description
BRIEF DESCRIPTION OF DRAWINGS
[0054]
[0055]
[0056]
[0057]
[0058]
[0059]
[0060]
[0061]
[0062]
[0063]
[0064]
[0065]
[0066]
[0067]
[0068]
[0069]
[0070]
[0071]
[0072]
[0073]
[0074]
[0075]
[0076]
[0077]
[0078]
[0079]
[0080]
[0081]
[0082]
[0083]
[0084]
[0085]
[0086]
[0087]
DETAILED DESCRIPTION OF EMBODIMENTS
[0088] Hereinafter, the present invention is described in detail with reference to specific implementations and examples, and the advantages and various effects of the present invention may be more clearly presented therefrom. It should be understood by those skilled in the art that these specific implementations and examples are intended to illustrate the present invention, instead of limiting the present invention.
Example 1. Primers and Probes Used in the Present Invention
[0089] Primers and probes used in the present invention are as shown below:
TABLE-US-00008 8782 C-type forward primer: (SEQ ID NO: 1) 5′-GCTGATTTTGACACATGGTTCATC-3′; 28144 T-type forward primer: (SEQ ID NO: 2) 5′-ATCGGTAATTATACAGTTTCCAGATT-3′; 8782 T-type forward primer: (SEQ ID NO: 3) 5′-GCTGATTTTGACACATGGTATCGT-3′; 28144 C-type forward primer: (SEQ ID NO: 4) 5′-ATCGGTAATTATACAGTTTCCTTTGC-3′; 8782 reverse primer: (SEQ ID NO: 5) 5′-CCATTAGTTGTGCGTAATATCGT-3′. 8782 probe: (SEQ ID NO: 6) 5′-CTTGCCCATTGATTGCTGCAGTCATAA-3′; 28144 reverse primer: (SEQ ID NO: 7) 5′-CAACACGAACGTCATGATACTCTA-3′; and 28144 probe: (SEQ ID NO: 8) 5′-ATTGCCAGGAACCTAAATTGGGTAGT-3′.
[0090] The fluorescent reporter group of the 8782 probe is HEX, and the fluorescent reporter group of the 28144 probe is FAM. The 3′ end of the probe also has a quenching group of BHQ1 or BHQ2.
Example 2. Method for Detecting Mutations of 2019 Novel Coronavirus
[0091] A sample for detection in the present invention is a throat swab, sputum, bronchoalveolar lavage fluid, or blood. The following operations are carried out:
[0092] (1) mixing a nucleic acid release agent S1014 of Sansure Biotech with a positive control at a ratio of 1:1, and lysing at room temperature for 10 minutes;
[0093] (2) according to the proportions of 26 μL of each reaction solution and 4 μL of mixed enzyme, taking a corresponding amount of reaction solution and mixed enzyme, mixing well, and dispensing 30 μL of each reaction into each reaction tube; adding 20 μL of the lysed to-be-tested sample to each reaction tube, then covering with a PCR tube cap, instantaneously centrifuging, and placing it in a real-time fluorescent PCR instrument; and
[0094] (3) performing reaction and detection under the following cycle conditions (a FAM channel and a HEX channel are selected for fluorescence acquisition):
TABLE-US-00009 Step Temperature Time Cycle number Reverse transcription stage 50° C. 30 minutes 1 cDNA pre-denaturation 95° C. 1 minute 1 Denaturation 95° C. 15 seconds 45 Annealing, extension and 60° C. 30 seconds fluorescence acquisition
[0095] Result Analysis:
[0096] 1) Target detection signals are FAM and HEX.
[0097] 2) Baseline setting: the Baseline is generally set as 3-15 cycles, and may be adjusted according to actual situations. The adjusting principle is: selecting a region with relatively stable fluorescence signal before exponential amplification, the start avoiding signal fluctuation in an initial stage of fluorescence collection, and the end being 1-2 cycles less than the sample Ct with the earliest exponential amplification. Threshold setting: the setting principle is that a threshold line just exceeds the highest point of a normal negative control.
[0098] 3) If there is no amplification signal in each channel of the two compositions, the sample concentration is lower than the detection lower limit.
[0099] If in two compositions, one and only one composition has amplification signals:
[0100] FAM and HEX of the first composition both have an amplification curve, and Ct<40, representing C8782 and T28144 mutations of the 2019 novel coronavirus.
[0101] FAM and HEX of the second composition both have an amplification curve, and Ct<40, representing T8782 and C28144 mutations of the 2019 novel coronavirus.
[0102] If an amplification signal exists in both of the two compositions, the difference between the Ct values of the corresponding channels in the two compositions are compared.
[0103] The difference of Ct values of the channels in the two compositions is greater than 10. If the Ct value of the first composition is relatively small, it is C8782 and T28144 mutations of the 2019 novel coronavirus. If the Ct value of the second composition is relatively small, it is T8782 and C28144 mutations of the 2019 novel coronavirus.
[0104] If the difference of Ct values of the channels in the two compositions is less than 10, the two types exist simultaneously.
[0105] For the first composition, when the FAM and HEX both have an amplification curve, and Ct<35, it represents C8782 and T28144 mutations of the 2019 novel coronavirus.
[0106] For the second composition, when the FAM and HEX both have an amplification curve, and Ct<35, it represents T8782 and C28144 mutations of the 2019 novel coronavirus.
[0107] For the first composition and the second composition, if the FAM and HEX have only one or no amplification curve, the type of the 2019 novel coronavirus cannot be determined. The determining result is as shown in Table 1 below.
[0108] The results of amplification in the two compositions are analyzed, where “+” refers to an amplification curve or Ct value less than 45, and “-” refers to no amplification curve or Ct value greater than 45. Ct.sub.1 and Ct.sub.2 are the Ct values of the corresponding channels in the first composition and the second composition, respectively; ΔCt is an absolute value of the difference between the two Ct values.
TABLE-US-00010 TABLE 1 First Second Channel composition composition Ct value Result FAM − − Low viral load + − 28144 T-type − + 28144 C-type + + ΔCt ≥ 10, and 28144 T-type Ct1 < Ct2 ΔCt ≥ 10, and 28144 C-type Ct1 > Ct2 ΔCt < 10 28144 C/T hybrid-type HEX − − Low viral load + − 8782 C-type − + 8782 T-type + + ΔCt ≥ 10, and 8782 C-type Ct1 < Ct2 ΔCt ≥ 10, and 8782 T-type Ct1 > Ct2 ΔCt < 10 8782 C/T hybrid-type
Example 3. Composition of the Present Invention for Detecting Mutations of the 2019 Novel Coronavirus
[0109] Detection is made using the composition in Example 1 of the present invention according to the method in Example 2. The sample is a nucleic acid extracted from clinically positive samples. The first composition and the second composition are respectively used for detection, and results thereof are as shown in
Example 4. Specificity Detection of the Composition of the Present Invention
[0110] Detection is made using the composition in Example 1 of the present invention according to the method in Example 2. The samples are two pseudovirus samples with high concentration (the concentration is 1.0E+08 copy/mL). The first composition and the second composition are respectively used for detecting C8782 and T28144 mutations of the 2019 novel coronavirus and the pseudovirus templates of T8782 and C28144 mutations of the 2019 novel coronavirus. The results are as shown in
TABLE-US-00011 TABLE 2 T8782 and C28144 Pseudovirus templates Pseudovirus templates mutations of the of C8782 and T28144 of T8782 and C28144 2019 novel mutations of the 2019 mutations of the 2019 coronavirus novel coronavirus novel coronavirus First composition 23.09 37.06 FAM Second composition 36.13 23.48 FAM ΔCt-FAM 13.04 13.58 First composition 23.44 37.69 HEX Second composition 36.44 23.49 HEX ΔCt-HEX 13.00 14.20
[0111] In view of the above, there are significant differences in amplification efficiency of different compositions against C8782 and T28144 mutation pseudoviruses of the 2019 novel coronavirus, and T8782 and C28144 mutant pseudoviruses of the 2019 novel coronavirus. The minimum value of each ΔCt (13.00) is still much greater than the requirement (10) of ΔCt in the result determination method in Example 2.
[0112] Calculation is conducted based on the minimum value of ΔCt of 13.00 in each group, the amplification efficiency difference between the two compositions for the specific template and non-specific template is up to 8192 times (2{circumflex over ( )}13), indicating excellent specificity.
Example 5. Precision Detection of the Composition of the Present Invention
[0113] Detection is made using the composition in Example 1 of the present invention according to the method in Example 2. The samples are pseudovirus samples (C8782 and T28144 mutation pseudoviruses of the 2019 novel coronavirus, and T8782 and C28144 mutation pseudoviruses of the 2019 novel coronavirus) which are diluted by normal saline. Each concentration gradient is separately 1.0E+06 copy/mL, 1.0E+05 copy/mL, 1.0E+04 copy/mL, 1.0E+03 copy/mL, 1.0E+02 copy/mL, and 1.0E+01 copy/mL. The first composition and the second composition are respectively used for detection of specific templates with different concentrations, and results thereof are as shown in
[0114] The lowest detection concentration of the first composition is 1.0E+03 copy/mL (10 copy/reaction).
[0115] The lowest detection concentration of the second composition is 1.0E+03 copy/mL (10 copy/reaction).
Comparison Example 1. Other Probe and Primer Compositions of the Present Invention for Detecting Mutations of the 2019 Novel Coronavirus
[0116] In the creation process of the present invention, dozens of different specific recognition primers are designed for each site base. These primers all follow the design principle of ARMS primers, 1-2 mutations are added at the 3′ end to selectively amplify specific targets. The sequences of some comparison primers are as follows:
TABLE-US-00012 8782 C-type forward comparison primers: 8782 C-1: (SEQ ID NO: 11) 5′-GCTGATTTTGACACATGGTTTAGC-3′; 8782 C-2: (SEQ ID NO: 12) 5′-GCTGATTTTGACACATGGTTTATC-3′; 8782 C-3: (SEQ ID NO: 13) 5′-GCTGATTTTGACACATGGTTTCGC-3′; 8782 C-4: (SEQ ID NO: 14) 5′-GCTGATTTTGACACATGGTTTCTC-3′; and 8782 C-5: (SEQ ID NO: 15) 5′-GCTGATTTTGACACATGGTCTCGT-3′; 8782 T-type forward comparison primers: 8782 T-1: (SEQ ID NO: 16) 5′-GCTGATTTTGACACATGGTTTAGT-3′; 8782 T-2: (SEQ ID NO: 17) 5′-GCTGATTTTGACACATGGTTTCGT-3′; 8782 T-3: (SEQ ID NO: 18) 5′-GCTGATTTTGACACATGGTTTATT-3′; 8782 T-4: (SEQ ID NO: 19) 5′-GCTGATTTTGACACATGGTTTCAT-3′; and 8782 T-5: (SEQ ID NO: 20) 5′-GCTGATTTTGACACATGGTTGATT-3′; 28144 T-type forward comparison primers: 28144 T-1: (SEQ ID NO: 21) 5′-ATCGGTAATTATACAGTTTCCTGTTT-3′; 28144 T-2: (SEQ ID NO: 22) 5′-ATCGGTAATTATACAGTTTCCTCTTT-3′; 28144 T-3: (SEQ ID NO: 23) 5′-ATCGGTAATTATACAGTTTCCTGGTT-3′; 28144 T-4: (SEQ ID NO: 24) 5′-ATCGGTAATTATACAGTTTCCTGGGT-3′; and 28144 T-5: (SEQ ID NO: 25) 5′-ATCGGTAATTATACAGTTTCCTCTCT-3′; and 28144 C-type forward comparison primers: 28144 C-1: (SEQ ID NO: 26) 5′-ATCGGTAATTATACAGTTTCCTGTTC-3′; 28144 C-2: (SEQ ID NO: 27) 5′-ATCGGTAATTATACAGTTTCCTCTTC-3′; 28144 C-3: (SEQ ID NO: 28) 5′-ATCGGTAATTATACAGTTTCCTGGTC-3′; 28144 C-4: (SEQ ID NO: 29) 5′-ATCGGTAATTATACAGTTTCCTGAGC-3′; 28144 C-5: (SEQ ID NO: 30) 5′-ATCGGTAATTATACAGTTTCCAGGTC-3′.
[0117] These primers are combined with other primers and probes in Example 1 to form compositions, and the method in Embodiment 2 is used for detection. The samples are two pseudovirus samples with the concentration of 1.0E+08 copy/mL. The test results are as shown in
[0118] Main indexes to evaluate the quality of the primers are the amplification efficiency and specificity of the primer. The smaller the Ct value of the primer for detection and amplification specific target is, the higher the amplification efficiency thereof is. When the Ct values of the specific template and the non-specific template at the same concentration are detected by the primer, the absolute value of the difference between the Ct values of the two templates is as large as possible, and the specificity thereof is better.
[0119] It can be seen from each result that the overall performances of the amplification efficiency and specificity of the comparative primers are inferior to those of the composition of the present invention.
REFERENCE TO AN ELECTRONIC SEQUENCE LISTING
[0120] The contents of the electronic sequence listing (CU694SequenceListing.xml; Size: 34,281 bytes; and Date of Creation: Aug. 17, 2022) is herein incorporated by reference in its entirety.