CN111676322A - Primer composition, kit, method and protective case for 7 coronavirus typing - Google Patents
Primer composition, kit, method and protective case for 7 coronavirus typing Download PDFInfo
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Abstract
The invention discloses a primer composition, a kit, a method and a protective case for 7 kinds of coronavirus (HCoV-229E, HCoV-HKU1, MERS-CoV, HCoV-NL63, HCoV-OC43, SARS-CoV and SARS-CoV-2) typing. The primer composition, the kit and the method have high detection specificity, the sensitivity meets the standard, the detection time is 45 minutes, a positive sample can show a signal within 20 minutes, the chip is verified by using a negative and positive sample stored in a laboratory, and the effect is ideal. In addition, equipment such as a detection chip, an isothermal amplification instrument, a signal scanner, a centrifugal machine and the like are integrated in a protective box, the limitation of the existing detection technology to personnel/places is broken through, the convenience degree is improved when the detection is shortened, the diagnosis is pushed to move forward, an on-site rapid detection means is provided for suspected patients and close contact people on the port site, and the prevention and control pressure of the current port on the input novel coronavirus infection cases is relieved.
Description
Technical Field
The invention relates to the field of coronavirus detection, in particular to a primer composition, a kit, a method and a protective case for 7 coronavirus typing.
Background
Coronaviruses (Coronaviruses) are RNA viruses belonging to the order nonsegmentoviridae, the largest positive-strand RNA viruses known today. The following 7 types of coronaviruses currently known to cause human infection are available: HCoV-229E, HCoV-HKU1, MERS-CoV, HCoV-NL63, HCoV-OC43, SARS-CoV, and SARS-CoV-2. The novel coronavirus pneumonia caused by the novel coronavirus in 12 months in 2019 poses a significant and urgent threat to global public health, and a proper detection technology and a proper detection method are selected to accurately and quickly identify the etiology, so that the method plays a key role in improving the diagnosis and treatment efficiency of diseases and restraining the outbreak and prevalence of infectious diseases.
When a sample with unknown infection source is faced, the differential diagnosis of multiple coronaviruses is required to be carried out simultaneously, namely the parallel detection of multiple targets. Traditional pathogen detection methods are generally directed to multi-sample single-target detection; the detection process is complex, samples need to be transferred among different devices, the operation difficulty is high, and professionals are needed, and the requirements of pathogen integration, rapid and parallel detection cannot be met because the environment and resources of epidemic situation sites or primary hospitals are limited and corresponding experimental conditions are not usually provided.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a primer composition, a kit, a method and a protective box for 7 coronavirus typing, which are used for realizing the high-specificity and high-sensitivity parallel detection of multiple coronaviruses by integrating the processes of gene amplification, product detection and the like on one chip by utilizing a microfluidic chip technology.
In order to achieve the purpose, the invention adopts the following technical scheme:
the first aspect of the present invention provides a primer composition for typing 7 kinds of coronavirus, which comprises a primer having a sequence of SEQ ID NO: 1 to SEQ ID NO: 5, and the sequence of the primer group is SEQ ID NO: 7 to SEQ ID NO: 11, HCoV-HKU1 amplification primer set having the sequence of SEQ ID NO: 13 to SEQ ID NO: 17, the MERS-CoV amplification primer group has a sequence of SEQ ID NO: 19 to SEQ ID NO: 23, HCoV-NL63 amplification primer set having the sequence of SEQ ID NO: 25 to SEQ ID NO: 29, and the sequence of the HCoV-OC43 amplification primer group is SEQ ID NO: 31 to SEQ ID NO: 35 and the sequence is SEQ ID NO: 37 to SEQ ID NO: 42 or SEQ ID NO: 44-SEQ ID NO: 48 of the SARS-CoV-2 amplification primer set.
The second aspect of the invention provides a kit for 7 kinds of coronavirus typing, which comprises fluorescent isothermal amplification premix (buffer for short), negative control, a microfluidic chip and a sealing film;
each reaction well of the microfluidic chip comprises a primer embedding system containing a primer solution of any one of the amplification primer sets in the primer composition as in the first aspect;
the 7 kinds of coronavirus include HCoV-229E, HCoV-HKU1, MERS-CoV, HCoV-NL63, HCoV-OC43, SARS-CoV and SARS-CoV-2.
Furthermore, the primer embedding system also comprises a trehalose solution with the concentration of 0.5 percent and water.
A third aspect of the present invention is to provide a method for typing 7 coronaviruses using the kit of the second aspect, comprising the steps of:
step one, preparing a sample to be detected: extracting virus nucleic acid RNA in a sample by adopting an RNA extraction kit to serve as a sample to be detected;
mixing the fluorescent isothermal amplification premix solution with a sample to be detected or a negative control, then carrying out vortex oscillation and uniform mixing, and carrying out instantaneous centrifugation to obtain a mixed solution;
adding the mixed solution into a sample adding hole of the micro-fluidic chip, and sealing the sample adding hole and the air vent by using a sealing film; removing bubbles by using a scraping blade to ensure that the sealing film is completely attached;
putting the microfluidic chip into a microfluidic constant-temperature amplification instrument, setting reaction conditions and an operation program, and performing amplification; and after the amplification is finished, analyzing the result finally.
Further, the volume ratio of the fluorescent isothermal amplification premix liquid to the sample to be detected in the second step is 5: 1.
Further, the reaction conditions in the fourth step were 65 ℃ and the reaction time was 45 min.
Further, the operation procedure in the fourth step is as follows: low-speed centrifugal rotation speed: 1600r/min, low speed centrifugation time: 30 sec; high-speed centrifugal rotation speed: 4600r/min, high speed centrifugation time: for 30 sec.
A fourth aspect of the invention provides a protective case comprising the kit of the second aspect, the protective case further comprising an isothermal amplification apparatus, a signal scanner, and a centrifuge.
By adopting the technical scheme, compared with the prior art, the invention has the following technical effects:
(1) fast and efficient: the whole amplification can be completed within 15-30 min, and the amplification yield can reach 109-1010A copy;
(2) the operation is simple and convenient: complex instruments, special reagents, complicated steps such as denaturation of double-stranded DNA and the like are not needed in advance, reaction and detection can be carried out by only one real-time fluorescence detector, and the conditions are mild;
(3) specificity: no cross reaction with other pathogens;
(3) high sensitivity: the minimum detection limit can reach 100 copies;
(4) multiple typing: the kit can synchronously analyze 7 human coronaviruses at one time;
in conclusion, the invention provides a primer composition, a kit, a method and a protective case for 7 coronavirus typing. The primer composition, the kit and the method have high detection specificity, the sensitivity meets the standard, the detection time is 45 minutes, a positive sample can show a signal within 20 minutes, the chip is verified by using a negative and positive sample stored in a laboratory, and the effect is ideal. In addition, equipment such as a detection chip, an isothermal amplification instrument, a signal scanner, a centrifugal machine and the like are integrated in a protective box, the limitation of the existing detection technology to personnel/places is broken through, the convenience degree is improved when the detection is shortened, the diagnosis is pushed to move forward, an on-site rapid detection means is provided for suspected patients and close contact people on the port site, and the prevention and control pressure of the current port on the input novel coronavirus infection cases is relieved.
Drawings
FIG. 1 is a graph showing the amplification of ORF1ab gene in cDNA samples of different concentrations in accordance with one embodiment of the present invention;
FIG. 2 is a graph showing the amplification of N gene in cDNA samples of different concentrations according to an embodiment of the present invention;
FIG. 3 is a graph showing the amplification curves of different concentrations of HCoV-229E cDNA plasmid in one embodiment of the present invention, in which FIG. A shows HCoV-229E cDNA stock solution, and FIG. B shows HCoV-229E cDNA stock solution × 10-1Doubling; panel C shows no amplification of HCoV-229E cDNA from subsequent dilution to negative control water;
FIG. 4 is a graph showing the amplification of cDNA plasmids HCoV-HKU1 at different concentrations in one embodiment of the present invention, wherein FIG. A shows the original solution of cDNA HCoV-HKU1, and FIG. B shows the original solution × 10 of cDNA of HCoV-HKU1-1Doubling; panel C shows that none of the HCoV-HKU1 cDNA was amplified from subsequent dilution to negative control water;
FIG. 5 is a graph showing the amplification curves of different concentrations of MERS-CoV cDNA plasmid in one embodiment of the present invention; the concentrations of panels A-E are 10 for MERS-CoV cDNA stock solutions, respectively0、10-1、10-2、10-3、10-4Fold, panel F is no amplification of MERS-CoV cDNA from subsequent dilution fold to negative control water;
FIG. 6 is a graph of the amplification of different concentrations of HCoV-NL63 cDNA plasmid in one embodiment of the invention; FIGS. A-D show the concentrations of 10 of HCoV-NL63 cDNA stock solutions, respectively0、10-1、10-2、10-3Fold, panel E shows no amplification of HCoV-NL63 cDNA from subsequent dilution to negative control water;
FIG. 7 shows different concentrations of H in an embodiment of the present inventionAmplification profile of the CoV-OC43 cDNA plasmid; the concentrations in panels A-D are 10 of HCoV-OC43 cDNA stock solutions, respectively0、10-1、10-2、10-3Fold, panel E shows that none of the HCoV-OC43 cDNA was amplified after subsequent dilution to negative control water;
FIG. 8 is a graph of the amplification curves of different concentrations of SARS-CoV cDNA plasmid in one embodiment of the invention; the concentration of SARS plasmid in each of the graphs A to G was 107、106、105、104、103、102、101copies/. mu.L, panel H is a negative control.
Detailed Description
The invention provides a primer composition, a kit, a method and a protective case for typing 7 kinds of coronavirus, wherein the 7 kinds of coronavirus comprise HCoV-229E, HCoV-HKU1, MERS-CoV, HCoV-NL63, HCoV-OC43, SARS-CoV and SARS-CoV-2.
The present invention will be described in detail and specifically with reference to the following examples to facilitate better understanding of the present invention, but the following examples do not limit the scope of the present invention.
Example 1
This example provides primer compositions and kits for typing of 7 coronaviruses.
Primer composition
Specific LAMP primers are designed aiming at 7 human coronavirus conservative fragments, and a primer composition which is high in specificity and used for 7 coronavirus types is screened out through a large number of primer primary screening, loop primer screening and specificity verification experiments.
HCoV-229E primer sequences and target sequences
HCoV-HKU1 primer sequence and target sequence
MERS-CoV primer sequence and target sequence
HCoV-NL63 primer sequence and target sequence
HCoV-OC43 primer sequence and target sequence
SARS-CoV primer sequence and target sequence
Primer sequence and target sequence of SARS-CoV-2
Specific LAMP primers are designed aiming at ORF1ab and N gene of SARS-CoV-2, and the screened primer sequences with strong specificity are as follows:
in addition, in the primer screening process, each primer did not cross-react with the negative control, water, and it was verified that the primer set of one specific coronavirus did not cross-react with the plasmids of other coronaviruses. Therefore, any one of the primer sets in the primer composition has good specificity.
Second, kit
A kit for typing 7 kinds of coronavirus, which comprises the primer composition, and comprises the following components:
wherein, the detection chip is a microfluidic chip, and each reaction hole comprises a primer embedding system containing a primer solution of any one amplification primer group in the primer composition; the primer embedding system also comprises a trehalose solution with the concentration of 0.5%, water and an internal standard substance.
Example 2
This example provides a method for typing 7 coronaviruses using the primer composition and kit of example 1.
1. Experimental methods
1.1 sample pretreatment
1.1.1 nasopharyngeal swab samples are placed in sample storage tubes, numbered and sent to the laboratory.
1.2 storage and transportation of samples
The collected or processed sample is preserved for no more than 24 hours at the temperature of 2-8 ℃; if the product is stored for a long time, the product should be placed in a refrigerator at-70 ℃ to avoid repeated freeze thawing (the freeze thawing time is not more than 3 times). After the collected sample is sealed, a thermal insulation kettle or a thermal insulation barrel is adopted to be sealed by adding ice, and the sample is sent to a laboratory within 6-8 hours.
1.3 template preparation (sample preparation area)
Viral nucleic acid RNA in various samples extracted by adopting an RNA extraction kit is used as a template to be detected and is stored at minus 80 ℃. It is recommended to use 200. mu.L of liquid sample for nucleic acid extraction. The nucleic acid extraction reagent (Shanghai Kagaku Kogyo 20170099) can be used.
1.4 reagent preparation (reagent preparation area)
The kit is taken out from minus 20 +/-5 ℃, and each reagent is unfrozen at room temperature, fully mixed and centrifuged for a short time for use. Taking N (N is the number of samples to be detected) 1.5mL centrifuge tubes, adding 60 mu L of fluorescent isothermal amplification premix into each tube, arranging a negative control hole on each chip, and taking the negative control as sample loading.
1.5 sample application
One detection chip can detect 4 samples to be detected simultaneously.
Adding 12 mu L of sample nucleic acid templates to be detected into a centrifuge tube prepared with reagents, mixing uniformly by vortex oscillation, performing instantaneous centrifugation, adding all the mixed solution into corresponding sample adding holes of a detection chip (the sample adding system on each sample adding hole is 72 mu L), and sealing the sample adding holes and the air holes by a chip sealing film. And (4) removing bubbles by using a scraping blade to ensure that the sealing film is completely attached.
1.6 constant temperature amplification (amplification zone)
Starting a micro-fluidic constant-temperature amplification instrument, selecting a 4-hole chip, and carrying out reaction conditions: the temperature was set to 65 ℃ and the reaction time was set to 45min, and the program was run (low-speed centrifugation speed: 1600r/min, low-speed centrifugation time: 30 sec; high-speed centrifugation speed: 4600r/min, high-speed centrifugation time: 30 sec).
1.7 analysis of results
The threshold line is set to 800 (which can be adjusted according to the actual situation, the threshold line just exceeds the highest point of the atypical S-type amplification curve according to the set principle, and the Ct value is displayed as 0), and the instrument matching software automatically analyzes the result.
2. Method for determining result
2.1 Positive: within 45 minutes of reaction time, an obvious S-shaped amplification curve appears at any detection hole site, and the corresponding detection item of the hole is judged to be positive.
2.2 negative: and within 45 minutes of reaction time, detecting that no amplification curve exists in the hole position, and judging that the corresponding detection item is negative.
Wherein, aiming at SARS-CoV-2, when the detection hole of ORF1ab gene and the detection hole of N gene are simultaneously positive, the sample is judged to be SARS-CoV-2 positive;
if only one of the wells is positive, the experiment needs to be repeated;
if the sample is still positive in one hole and negative in the other hole after the repeated experiment, the sample is judged to be SARS-CoV-2 suspected positive and needs further rechecking;
when both wells are negative, the sample can be judged to be SARS-CoV-2 negative.
Verification example 1
This example was conducted to investigate the sensitivity of the primer combinations screened in example 1, using 10-fold gradient dilution of cDNA samples, to test their sensitivity. Wherein, when in detection, aiming at SARS-CoV-2cDNA plasmid, samples are respectively diluted by 10 times, 100 times, 1000 times and 10000 times; for SARS-CoV cDNA plasmid, samples were diluted 10110 times of210 times of310 times of410 times of510 times of6Multiple sum of 107And (4) doubling. The results of the tests are shown in FIGS. 1 to 8.
As can be seen from FIGS. 1-2, the primer set for ORF1ab gene was able to detect 1000-fold dilution of the cDNA stock solution and the primer set for N gene was able to detect 1000-fold dilution of the cDNA stock solution against the SARS-CoV-2 plasmid.
As can be seen from FIG. 3, the primer set of HCoV-229E detected a 10-fold dilution of the cDNA stock.
As can be seen from FIG. 4, the primer set of HCoV-HKU1 could detect 10-fold dilution of the cDNA stock solution.
As shown in FIG. 5, the primer set of MERS-CoV can detect 10000-fold dilution of the cDNA stock solution, but only one curve is generated in the duplicate wells.
As can be seen from FIG. 6, the primer set of HCoV-NL63 was found to have irregular peaks in 10-fold dilution of the cDNA stock solution and subsequent dilution.
As can be seen from FIG. 7, the primer set of HCoV-OC43 can detect 100-fold dilution of cDNA stock solution, and the peak is atypical due to subsequent dilution.
As can be seen from FIG. 8, SARS-CoV was detected by diluting the cDNA stock solution 10 times.
The embodiments of the present invention have been described in detail, but the embodiments are merely examples, and the present invention is not limited to the embodiments described above. Any equivalent modifications and substitutions to those skilled in the art are also within the scope of the present invention. Accordingly, equivalent changes and modifications made without departing from the spirit and scope of the present invention should be covered by the present invention.
Sequence listing
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Qingdao International Travel Health Care Center (Qingdao customs port clinic)
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<213>Artificial Sequence
<400>34
attggcatca ttaacaacac agtttttgaa gtacttgtcc agct 44
<210>35
<211>21
<212>DNA
<213>Artificial Sequence
<400>35
atgatcctct gcaacctgag c 21
<210>36
<211>621
<212>DNA
<213>Artificial Sequence
<400>36
gtcacgtatg tgccatccca ggagaggaac ttcaccacag cgccagcaat ttgtcatgaa 60
ggcaaagcat acttccctcg tgaaggtgtt tttgtgttta atggcacttc ttggtttatt 120
acacagagga acttcttttc tccacaaata attactacag acaatacatt tgtctcagga 180
aattgtgatg tcgttattgg catcattaac aacacagttt atgatcctct gcaacctgag 240
cttgactcat tcaaagaaga gctggacaag tacttcaaaa atcatacatc accagatgtt 300
gatcttggcg acatttcagg cattaacgct tctgtcgtca acattcaaaa agaaattgac 360
cgcctcaatg aggtcgctaa aaatttaaat gaatcactca ttgaccttca agaattggga 420
aaatatgagc aatatattaa atggccttgg tatgtttggc tcggcttcat tgctggacta 480
attgccatcg tcatggttac aatcttgctt tgttgcatga ctagttgttg cagttgcctc 540
aagggtgcat gctcttgtgg ttcttgctgc aagtttgatg aggatgactc tgagccagtt 600
ctcaagggtg tcaaattaca t621
<210>37
<211>22
<212>DNA
<213>Artificial Sequence
<400>37
acttaaaaac acagtctgta cc 22
<210>38
<211>19
<212>DNA
<213>Artificial Sequence
<400>38
tcaaaagccc tgtatacga 19
<210>39
<211>39
<212>DNA
<213>Artificial Sequence
<400>39
tgactgaagc atgggttcgc gtctgcggta tgtggaaag 39
<210>40
<211>44
<212>DNA
<213>Artificial Sequence
<400>40
gctgatgcac aatcgttttt aaacgcatca gtactagtgc ctgt 44
<210>41
<211>25
<212>DNA
<213>Artificial Sequence
<400>41
agttgatcac aactacagcc ataac 25
<210>42
<211>19
<212>DNA
<213>Artificial Sequence
<400>42
ggtttgcggt gtaagtgca 19
<210>43
<211>329
<212>DNA
<213>Artificial Sequence
<400>43
tttacactta aaaacacagt ctgtaccgtc tgcggtatgt ggaaaggtta tggctgtagt 60
tgtgatcaac tccgcgaacc catgcttcag tcagctgatg cacaatcgtt tttaaacggg 120
tttgcggtgt aagtgcagcc cgtcttacac cgtgcggcac aggcactagt actgatgtcg 180
tatacagggc ttttgacatc tacaatgata aagtagctgg ttttgctaaa ttcctaaaaa 240
ctaattgttg tcgcttccaa gaaaaggacg aagatgacaa tttaattgat tcttactttg 300
tagttaagag acacactttc tctaactac 329
<210>44
<211>19
<212>DNA
<213>Artificial Sequence
<400>44
ggcagtcaag cctcttctc 19
<210>45
<211>20
<212>DNA
<213>Artificial Sequence
<400>45
<210>46
<211>40
<212>DNA
<213>Artificial Sequence
<400>46
ttcccctact gctgcctgga gttcctcatc acgtagtcgc 40
<210>47
<211>42
<212>DNA
<213>Artificial Sequence
<400>47
ttctcctgct agaatggctg gctctgtcaa gcagcagcaa ag 42
<210>48
<211>21
<212>DNA
<213>Artificial Sequence
<400>48
aatggcggtg atgctgctct t 21
<210>49
<211>340
<212>DNA
<213>Artificial Sequence
<400>49
gcggcagtca agcctcttct cgttcctcat cacgtagtcg caacagttca agaaattcaa 60
ctccaggcag cagtagggga acttctcctg ctagaatggc tggcaatggc ggtgatgctg 120
ctcttgcttt gctgctgctt gacagattga accagcttga gagcaaaatg tctggtaaag 180
gccaacaaca acaaggccaa actgtcacta agaaatctgc tgctgaggct tctaagaagc 240
ctcggcaaaa acgtactgcc actaaagcat acaatgtaac acaagctttc ggcagacgtg 300
gtccagaaca aacccaagga aattttgggg accaggaact 340
Claims (8)
1. A primer composition for typing 7 kinds of coronaviruses, comprising a primer having the sequence of SEQ ID NO: 1 to SEQ ID NO: 5, and the sequence of the primer group is SEQ ID NO: 7 to SEQ ID NO: 11, HCoV-HKU1 amplification primer set having the sequence of SEQ ID NO: 13 to SEQ ID NO: 17, the MERS-CoV amplification primer group has a sequence of SEQ ID NO: 19 to SEQ ID NO: 23, HCoV-NL63 amplification primer set having the sequence of SEQ ID NO: 25 to SEQ ID NO: 29, and the sequence of the HCoV-OC43 amplification primer group is SEQ ID NO: 31 to SEQ ID NO: 35 and the sequence is SEQ ID NO: 37 to SEQ ID NO: 42 or SEQ ID NO: 44-SEQ ID NO: 48 of the SARS-CoV-2 amplification primer set.
2. A kit for 7 kinds of coronavirus typing is characterized by comprising fluorescent isothermal amplification premix, negative control, a microfluidic chip and a sealing film;
each reaction well of the microfluidic chip comprises a primer embedding system containing a primer solution of any one of the amplification primer sets in the primer composition according to claim 1;
the 7 coronaviruses include HCoV-229E, HCoV-HKU1, MERS-CoV, HCoV-NL63, HCoV-OC43, SARS-CoV and SARS-CoV-2.
3. The kit of claim 2, wherein the primer embedding system further comprises a trehalose solution with a concentration of 0.5% and water.
4. A method for typing 7 kinds of coronaviruses using the kit according to claim 2 or 3, comprising the steps of:
step one, preparing a sample to be detected: extracting virus nucleic acid RNA in a sample by adopting an RNA extraction kit to serve as the sample to be detected;
mixing the fluorescent isothermal amplification premix solution with the sample to be detected or the negative control, then carrying out vortex oscillation and uniform mixing, and carrying out instantaneous centrifugation to obtain a mixed solution;
adding the mixed solution into a sample adding hole of the micro-fluidic chip, and sealing the sample adding hole and the air vent by using the sealing film; removing bubbles by using a scraping blade to ensure that the sealing film is completely attached;
putting the microfluidic chip into a microfluidic constant-temperature amplification instrument, setting reaction conditions and operation programs, and performing amplification; and after the amplification is finished, analyzing the result finally.
5. The method according to claim 4, wherein the volume ratio of the fluorescent isothermal amplification premix to the sample to be detected in the second step is 5: 1.
6. The method according to claim 4, wherein the reaction conditions in step four are 65 ℃ and the reaction time is 45 min.
7. The method according to claim 4, wherein the running procedure in step four is: low-speed centrifugal rotation speed: 1600r/min, low speed centrifugation time: 30 sec; high-speed centrifugal rotation speed: 4600r/min, high speed centrifugation time: for 30 sec.
8. A protective housing comprising the kit of claim 2 or 3, wherein the protective housing further comprises an isothermal amplification apparatus, a signal scanner, and a centrifuge.
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| CN112553380A (en) * | 2020-12-31 | 2021-03-26 | 哈尔滨星云医学检验所有限公司 | Method for rapidly detecting 12 respiratory viruses by utilizing multiplex PCR (polymerase chain reaction) technology and application thereof |
| CN113215231A (en) * | 2021-03-30 | 2021-08-06 | 成都里来生物科技有限公司 | Dual PCR detection method for SARS-CoV and COVID-19 virus |
| CN114317834A (en) * | 2022-02-25 | 2022-04-12 | 军事科学院军事医学研究院环境医学与作业医学研究所 | Kit and method for detecting new coronavirus |
| CN115044704A (en) * | 2021-11-22 | 2022-09-13 | 江汉大学 | MNP (MNP) marker locus of human coronavirus HCoV-229E, primer composition, kit and application of MNP marker locus |
| CN115044705B (en) * | 2021-11-22 | 2023-06-16 | 江汉大学 | MNP (MNP) marker locus of human coronavirus HCoV-NL63, primer composition, kit and application of MNP marker locus |
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| CN112553380A (en) * | 2020-12-31 | 2021-03-26 | 哈尔滨星云医学检验所有限公司 | Method for rapidly detecting 12 respiratory viruses by utilizing multiplex PCR (polymerase chain reaction) technology and application thereof |
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| CN113215231B (en) * | 2021-03-30 | 2022-08-05 | 成都里来生物科技有限公司 | Dual PCR detection method for SARS-CoV and COVID-19 virus |
| CN115044704A (en) * | 2021-11-22 | 2022-09-13 | 江汉大学 | MNP (MNP) marker locus of human coronavirus HCoV-229E, primer composition, kit and application of MNP marker locus |
| CN115044705B (en) * | 2021-11-22 | 2023-06-16 | 江汉大学 | MNP (MNP) marker locus of human coronavirus HCoV-NL63, primer composition, kit and application of MNP marker locus |
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| CN114317834A (en) * | 2022-02-25 | 2022-04-12 | 军事科学院军事医学研究院环境医学与作业医学研究所 | Kit and method for detecting new coronavirus |
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