CN112029902A - Primer group for simultaneously detecting seven human coronaviruses based on capillary electrophoresis and application thereof - Google Patents
Primer group for simultaneously detecting seven human coronaviruses based on capillary electrophoresis and application thereof Download PDFInfo
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Abstract
The invention relates to a primer group for simultaneously detecting seven human coronaviruses based on capillary electrophoresis and application thereof, belonging to the technical field of molecular biology. It provides a primer group for simultaneously detecting seven human coronaviruses, namely HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HCoV-HKUI, MERS-CoV and COVID-19 and application thereof. The invention can rapidly and simultaneously screen seven human coronaviruses of HCoV-OC43/HCoV-229E/SARS-CoV/HCoV-NL63/HCoV-HKU1/MERS-CoV/COVID-19, has the outstanding advantages of high detection sensitivity, strong specificity, capability of realizing multiple rapid and accurate detection, simple operation, convenient application and the like, and provides a feasible technical method for rapid detection of pathogens in the fields of clinical diagnosis, disease monitoring detection, inspection and quarantine and the like.
Description
Technical Field
The invention relates to a primer group for simultaneously detecting seven human coronaviruses based on capillary electrophoresis and application thereof, in particular to a specific primer group for detecting seven human coronaviruses based on capillary electrophoresis, and the specific primer group also relates to a molecular Marker for simultaneously detecting the seven human coronaviruses based on capillary electrophoresis, a sample to be detected, an RT-PCR reaction system, reaction conditions and result analysis, and belongs to the technical field of molecular biology.
Background
Coronaviruses are a large family of viruses widely found in nature and are commonly susceptible to infection by humans and other mammals and birds. Human coronavirus (Human Coronaviruses, hCoVs) is known to be seven kinds at present, Human coronavirus infection is one of important pathogens causing Human respiratory diseases, the detection rate of the coronavirus in common respiratory pathogens is 3% -10%, and the coronavirus is an important pathogen causing clinical symptoms such as cough, fever, laryngitis, bronchitis, pneumonia and the like caused by infection of infants, old people and adults with low immune function. Epidemiological research shows that four human coronary viruses, namely HCoV-HKUI, HCoV-229E, HCoV-NL63 and HCoV-OC43, are distributed globally, and clinical symptoms mainly include fever, cough, rhinitis, laryngitis, bronchitis, pneumonia and the like, so that the four human coronary viruses cause certain burden on the life health of human beings. SARS-CoV, MERS-CoV and COVID-19 can cause acute respiratory symptoms after infection of human, and severe patients cause respiratory system and renal function failure and death.
The early prevention and control are the precondition for preventing infectious diseases from spreading, guaranteeing public health safety and maintaining human life health, and the development of a rapid, sensitive and specific molecular detection technical method is the key of the early prevention and control and also provides important basis for guiding clinic and reasonably selecting antiviral drugs. At present, the detection technical method aiming at human coronavirus mainly comprises virus isolation culture, serological detection method, common PCR method, isothermal amplification method and fluorescent quantitative RT-PCR method. Virus isolation culture, serological detection method and common PCR method are more conventional detection methods, and the methods have the defects of low detection sensitivity, overlong detection time, high requirement on laboratory hardware, high technical requirement on operators and the like in the experimental process, and the detection timeliness and result accuracy cannot be ensured. Isothermal amplification and fluorescent quantitative RT-PCR are molecular detection techniques commonly used in most laboratories at present. The isothermal amplification method has the outstanding problems that detection equipment and detection reagents are expensive, cross contamination is easy to occur, the detection result is uncertain, and the like, and can only realize single pathogen detection, single-tube multiple detection and the like. The fluorescence quantitative RT-PCR method also faces the problem that only single-tube single-weight (at most two-weight) pathogen detection can be realized at present, so that the pathogen screening times are increased, the detection result time is greatly prolonged, the operation process is increased, the detection cost is greatly increased, and the rapid diagnosis and early prevention and control of diseases are not facilitated. The rapid and accurate detection of hCoVs not only has important significance for monitoring and preventing and controlling the prevalence of hCoVs, but also provides reliable laboratory basis for clinical early diagnosis, reasonable selection of antiviral drug treatment and the like.
Disclosure of Invention
The invention aims to overcome the defects of long time consumption, low detection efficiency, high detection cost, low detection timeliness and the like in the hCoVs detection process, provides a primer group for simultaneously detecting seven human coronaviruses based on capillary electrophoresis and application thereof, and has the outstanding advantages of high detection sensitivity, strong specificity, capability of realizing multiple rapid and accurate detection, simplicity in operation, convenience in application and the like.
According to the technical scheme, the conserved sequences of the individual coronaviruses are respectively searched according to seven human coronavirus genome sequences of HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HCoV-HKUI, MERS-CoV and COVID-19, and specific primer pairs suitable for PCR are respectively designed as the optimal sequences.
The method comprises the following steps of simultaneously detecting seven human coronavirus primer groups based on capillary electrophoresis, wherein the primer groups comprise seven pairs of primer pairs, namely a primer pair 1, a primer pair 2, a primer pair 3, a primer pair 4, a primer pair 5, a primer pair 6 and a primer pair 7;
the primer pair 1 is directed at human coronavirus HCoV-OC43, and specifically comprises an upstream primer F1, the sequence of which is shown as SEQ ID No. 1; a downstream primer R1, the sequence of which is shown in SEQ ID No. 2;
the primer pair 2 is specific to human coronavirus HCoV-229E and comprises an upstream primer F2, and the sequence is shown as SEQ ID No. 3; a downstream primer R2, the sequence of which is shown in SEQ ID No. 4;
the primer pair 3 is directed against human coronavirus SARS-CoV, and specifically comprises an upstream primer F3, the sequence of which is shown in SEQ ID No. 5; a downstream primer R3, the sequence of which is shown in SEQ ID No. 6;
the primer pair 4 is directed against human coronavirus HCoV-NL63, and specifically comprises an upstream primer F4, and the sequence is shown as SEQ ID No. 7; a downstream primer R4, the sequence of which is shown in SEQ ID No. 8;
the primer pair 5 is directed against human coronavirus HCoV-HKU1, and specifically comprises an upstream primer F5, the sequence of which is shown as SEQ ID No. 9; a downstream primer R5, the sequence of which is shown in SEQ ID No. 10;
the primer pair 6 is directed against the human coronavirus MERS-CoV, and specifically comprises an upstream primer F6, wherein the sequence is shown as SEQ ID No. 11; a downstream primer R6, the sequence of which is shown in SEQ ID No. 12;
the primer pair 7 is directed at the human coronavirus COVID-19 and specifically comprises an upstream primer F7, and the sequence is shown as SEQ ID No. 13; the sequence of the downstream primer R7 is shown as SEQ ID No. 14.
The primer pair 1, the primer pair 2, the primer pair 3, the primer pair 4, the primer pair 5, the primer pair 6 and the primer pair 7 are respectively used for specifically detecting the seven human coronaviruses, and the lengths of corresponding detection target fragments are respectively 122bp, 152bp, 107bp, 205bp, 162bp, 143bp and 99 bp.
Specifically, the results are shown in Table 1.
TABLE 1
The invention provides an RT-PCR amplification reaction system for simultaneously detecting seven human coronavirus RT-PCR amplification reaction systems, including HCoV-OC43/HCoV-229E/SARS-CoV/HCoV-NL63/HCoV-HKU1/MERS-CoV/COVID-19, and the reaction conditions and the detection steps are as follows:
(1) sample preparation: the sample nucleic acid can be directly extracted from nasopharynx type, alveolar lavage fluid, sputum and other samples, can be extracted manually or by a full-automatic nucleic acid extractor, and the extracted nucleic acid is directly used for the next amplification reaction.
(2) Preferably, the One-step qRT-PCR Kit (TOYOBO) is selected, and the reaction system of 25 μ L is taken as an example, and is specifically configured as follows, and the final concentration of the used amount is:
12.5 μ L of the reaction solution of Piper 2; 0.5 μ L of DNA polymerase; RT reverse transcriptase 0.5 μ L; upstream primer 1. mu.L 2.5. mu.M; downstream primer 1. mu.L 2.5. mu.M; 5 mu L of virus template RNA; ddH2O 4.5μL；Up to 25μL；
(3) Preferably, the PCR reaction procedure is shown in table 2 below:
TABLE 2
(4) Analyzing and judging results: the PCR amplification product was subjected to a full-automatic capillary electrophoresis apparatus (Qseq100, Yongding pot Biotechnology (Jiangsu) Ltd. (Su stock Co., Ltd. 20180095)), high-resolution clip S1(C105102) was selected, Alignment Marker MA1(C109100) was selected, Size Marker MA2(C109200) was selected, sample injection voltage was 4kv 10S, and separation pressure was 6kv 330S. The PCR amplification product was diluted 10-fold with Dilution Buffer (C104405) and subjected to capillary electrophoresis according to the instructions of the apparatus.
The invention has the beneficial effects that: the primer group and the application thereof provided by the invention have the outstanding advantages of high detection sensitivity, strong specificity, capability of realizing multiple rapid and accurate detection, simple operation, convenient application and the like, and provide a feasible technical method for rapid detection of pathogens in the fields of clinical diagnosis, disease monitoring detection, inspection and quarantine and the like.
Drawings
FIG. 1 example 1 is the signal map of capillary electrophoresis of seven human coronavirus.
FIG. 2 example 1 capillary electrophoresis gel imaging profile of seven human coronavirus
FIG. 3 is the establishment of seven human coronavirus capillary electrophoresis molecule markers.
FIG. 4HCoV-NL63/COVID-19/MERS-CoV capillary electrophoresis signal map.
FIG. 5 molecular marker judges the result of COVID-19 capillary electrophoresis.
FIG. 6 judgment of MERS-CoV capillary electrophoresis results by molecular marker.
FIG. 7 determination of capillary electrophoresis results of HCoV-NL63 by molecular marker.
Detailed Description
Example 1:
in the embodiment of the invention, the seven human coronavirus RT-PCR amplification reaction systems for simultaneously detecting HCoV-OC43/HCoV-229E/SARS-CoV/HCoV-NL63/HCoV-HKU1/MERS-CoV/COVID-19 provided by the invention have the following reaction conditions and detection steps:
(1) sample preparation: the sample nucleic acid can be directly extracted from nasopharynx type, alveolar lavage fluid, sputum and other samples, can be extracted manually or by a full-automatic nucleic acid extractor, and the extracted nucleic acid is directly used for the next amplification reaction.
(2) Preferably, the One-step qRT-PCR Kit (TOYOBO) is selected, and the reaction system of 25 μ L is taken as an example, and is specifically configured as follows, and the final concentration of the used amount is:
12.5 μ L of the reaction solution of Piper 2; 0.5 μ L of DNA polymerase; RT reverse transcriptase 0.5 μ L; upstream primer 1. mu.L 2.5. mu.M; downstream primer 1. mu.L 2.5. mu.M; 5 mu L of virus template RNA; ddH2O 4.5μL；Up to 25μL；
(3) Preferably, the PCR reaction procedure is shown in table 3 below:
TABLE 3
(4) Analyzing and judging results:
the PCR amplification product was subjected to a full-automatic capillary electrophoresis apparatus (Qseq100, Biotech, Inc. (Su Ji, Ltd., No. 20180095)), selected from high-resolution clips S1(C105102), Alignment Marker selected from MA1(C109100), Size Marker selected from MA2(C109200), sample injection voltage 4kv 10S, and separation pressure 6kv 330S. The PCR amplification product was diluted 10-fold with Dilution Buffer (C104405) and subjected to capillary electrophoresis according to the instructions of the apparatus.
The signal maps of the seven human coronavirus capillary electrophoresis are shown in figure 1. Wherein, the seven coronary PCR products can accurately judge out a single target fragment (the error is not more than 5bp) with expected size after capillary electrophoresis, and the method has better specificity, detection sensitivity and accuracy.
The imaging spectra of the capillary electrophoresis gel of seven human coronavirus are shown in FIG. 2. Wherein MA-2 is Size marker, A01-A07 present COVID-19, SARS-CoV, HCoV-OC43, MERS-CoV, HCoV-229E, HCoV-HKU1 and HCoV-NL 63. In FIG. 2, the PCR amplification products of seven human coronaviruses all amplified specific bands by electrophoresis.
The establishment of the seven human coronavirus capillary electrophoresis molecular marker is shown in FIG. 3. Because of the difference of the linear structures of different target genes, the deviation of the interpretation signal of the capillary electrophoresis target gene is not more than 5bp, according to the experimental data, the interpretation signal confidence intervals of the COVID-19 target gene and the SARS-CoV target gene are respectively set at 99 (+/-3) bp and 107 (+/-3) bp, and the interpretation signal confidence intervals of the other four human coronavirus target genes are all set at the target fragment +/-5 bp. By establishing the molecular marker for the coronavirus, accurate detection for seven human coronaviruses can be synchronously realized.
Example 2
The results of nucleic acid extraction, RT-PCR reaction system configuration and capillary electrophoresis analysis using HCoV-NL63 positive sample and COVID-19 and MERS-CoV pseudovirus particles as screening subjects and the established molecular marker as a reference as described in example 1 are shown in FIG. 4. In fig. 4, a molecular Marker is used as a reference, and the corresponding target fragment can be accurately detected after nucleic acid extraction, PCR amplification and electrophoresis, so that the purpose of accurately identifying coronavirus at the same time is achieved.
The judgment of the capillary electrophoresis result of COVID-19 by the molecular marker is shown in FIG. 5: the molecular marker identifies a Target fragment of the COVID-19, namely the size of the Target fragment is 99(+3/-3) bp (within a confidence interval), the size of the result fragment after capillary electrophoresis is 99bp, and the sample to be detected can be judged to be the detected COVID-19 virus nucleic acid within the confidence interval.
The judgment of the MERS-CoV capillary electrophoresis result by the molecular marker is shown in FIG. 6: the molecular marker identifies a Target fragment of MERS-CoV, namely the size of the Target fragment is 143(+5/-5) bp (within a confidence interval), the size of the result fragment is 142bp after capillary electrophoresis, and the sample to be detected can be judged to be detected MERS-CoV virus nucleic acid within the confidence interval.
As shown in FIG. 7, the capillary electrophoresis result of HCoV-NL63 by the molecular marker is judged, wherein the molecular marker identifies a Target fragment of NL63, namely the size of the Target fragment is 205(+5/-5) bp (within a confidence interval), the size of the result fragment is 201bp after capillary electrophoresis, and the sample to be detected can be judged to be the detected NL63 virus nucleic acid within the confidence interval.
The invention has unique innovation in the market, namely realizes simultaneous detection of seven human coronaviruses, has the outstanding advantages of accurate detection, strong specificity, high sensitivity, quick detection time and the like, and avoids the defects of long time consumption, high detection cost, easy cross contamination and the like caused by one-by-one detection. Similar related technologies and product reports are not found in the market at present, and the invention has wide application prospects in aspects of disease detection, infectious disease prevention and control, guidance of clinical treatment and the like.
The foregoing shows and describes the general principles, principal features, and advantages of the invention. It will be understood by those skilled in the art that the invention is not limited to the embodiments described above, which are merely illustrative of the principles of the invention, but that various changes and modifications may be made without departing from the spirit and scope of the invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.
Sequence listing
<110> Wuxi customs of the people's republic of China
Xinjiang international travel health care center (Wulu wood Qi customs port outpatient department)
Xinjiang Huanyuan environmental protection science and technology Limited company
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Claims (4)
1. The primer group for simultaneously detecting seven human coronavirus based on capillary electrophoresis is characterized in that: the primer group comprises seven pairs of primer pairs, namely a primer pair 1, a primer pair 2, a primer pair 3, a primer pair 4, a primer pair 5, a primer pair 6 and a primer pair 7;
the primer pair 1 is directed at human coronavirus HCoV-OC43, and specifically comprises an upstream primer F1, the sequence of which is shown as SEQ ID No. 1; a downstream primer R1, the sequence of which is shown in SEQ ID No. 2;
the primer pair 2 is specific to human coronavirus HCoV-229E and comprises an upstream primer F2, and the sequence is shown as SEQ ID No. 3; a downstream primer R2, the sequence of which is shown in SEQ ID No. 4;
the primer pair 3 is directed against human coronavirus SARS-CoV, and specifically comprises an upstream primer F3, the sequence of which is shown in SEQ ID No. 5; a downstream primer R3, the sequence of which is shown in SEQ ID No. 6;
the primer pair 4 is directed against human coronavirus HCoV-NL63, and specifically comprises an upstream primer F4, and the sequence is shown as SEQ ID No. 7; a downstream primer R4, the sequence of which is shown in SEQ ID No. 8;
the primer pair 5 is directed against human coronavirus HCoV-HKU1, and specifically comprises an upstream primer F5, the sequence of which is shown as SEQ ID No. 9; a downstream primer R5, the sequence of which is shown in SEQ ID No. 10;
the primer pair 6 is directed against the human coronavirus MERS-CoV, and specifically comprises an upstream primer F6, wherein the sequence is shown as SEQ ID No. 11; a downstream primer R6, the sequence of which is shown in SEQ ID No. 12;
the primer pair 7 is directed at the human coronavirus COVID-19 and specifically comprises an upstream primer F7, and the sequence is shown as SEQ ID No. 13; the sequence of the downstream primer R7 is shown as SEQ ID No. 14.
2. The primer set for simultaneously detecting seven human coronaviruses based on capillary electrophoresis as claimed in claim 1, which is characterized in that: the primer pair 1, the primer pair 2, the primer pair 3, the primer pair 4, the primer pair 5, the primer pair 6 and the primer pair 7 are respectively used for specifically detecting the seven human coronaviruses, and the lengths of corresponding detection target fragments are respectively 122bp, 152bp, 107bp, 205bp, 162bp, 143bp and 99 bp.
3. The application of the primer group for simultaneously detecting seven human coronaviruses based on capillary electrophoresis is characterized in that a detection kit is prepared by seven pairs of primers, and the seven human coronaviruses are simultaneously detected by RT-PCR amplification reaction.
4. The application of the primer group for simultaneously detecting seven human coronaviruses based on capillary electrophoresis as claimed in claim 3, wherein the RT-PCR reaction system and the reaction program are as follows:
the reaction system is exemplified by a 25. mu.L reaction system, and the final concentration of the used amount is specifically configured as follows: 12.5 μ L of the reaction solution of Piper 2; 0.5 μ L of DNA polymerase; RT reverse transcriptase 0.5 μ L; upstream primer 1. mu.L 2.5. mu.M; downstream primer 1. mu.L 2.5. mu.M; 5 mu L of virus template RNA; ddH2O 4.5μL；
The reaction procedure was as follows:
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| CN104342503A (en) * | 2014-10-29 | 2015-02-11 | 福建国际旅行卫生保健中心 | Method for simultaneously detecting twelve kinds of common respiratory viruses |
| CN110144422A (en) * | 2019-06-17 | 2019-08-20 | 中华人民共和国无锡海关 | The quadruple fluorescence quantitative detection kit of four kinds of human corona virus is detected simultaneously |
| CN111440897A (en) * | 2020-02-28 | 2020-07-24 | 江苏硕世生物科技股份有限公司 | Probe and primer composition for rapidly detecting seven coronaviruses and other respiratory pathogens |
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2020
- 2020-08-12 CN CN202010805717.9A patent/CN112029902A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104342503A (en) * | 2014-10-29 | 2015-02-11 | 福建国际旅行卫生保健中心 | Method for simultaneously detecting twelve kinds of common respiratory viruses |
| CN110144422A (en) * | 2019-06-17 | 2019-08-20 | 中华人民共和国无锡海关 | The quadruple fluorescence quantitative detection kit of four kinds of human corona virus is detected simultaneously |
| CN111440897A (en) * | 2020-02-28 | 2020-07-24 | 江苏硕世生物科技股份有限公司 | Probe and primer composition for rapidly detecting seven coronaviruses and other respiratory pathogens |
Non-Patent Citations (2)
| Title |
|---|
| MYUNGSUN PARK ET AL.: "Optimization of primer sets and detection protocols for SARS-CoV-2 of coronavirus disease 2019 (COVID-19) using PCR and real-time PCR", 《EXP MOL MED》 * |
| 牛培华等: "同时检测六种人类冠状病毒的单管多重RT-PCR方法的建立", 《中华预防医学杂志》 * |
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