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Fluorescent quantitative PCR detection method of human coronavirus and respiratory syncytial virus and application thereof

A human coronavirus, fluorescence quantitative technology, applied in the field of fluorescence quantitative PCR, to achieve good sensitivity and specificity, good reproducibility

Inactive Publication Date: 2020-01-24
INST OF PATHOGEN BIOLOGY CHINESE ACADEMY OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The traditional PCR method designs corresponding primers for each pathogen, and one reaction detects one pathogen, which is limited when multiple pathogens need to be detected

Method used

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  • Fluorescent quantitative PCR detection method of human coronavirus and respiratory syncytial virus and application thereof
  • Fluorescent quantitative PCR detection method of human coronavirus and respiratory syncytial virus and application thereof
  • Fluorescent quantitative PCR detection method of human coronavirus and respiratory syncytial virus and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Embodiment 1 fluorescent quantitative PCR method detects human coronavirus OC43

[0029] 1.1 Extraction of nucleic acid

[0030] Take clinical samples from patients with acute respiratory infection, such as sputum, nasopharyngeal swabs, alveolar lavage fluid, etc., and extract nucleic acids according to conventional methods. The obtained nucleic acid was dissolved in 50 μl of eluent, and the extracted nucleic acid was quantified and tested for purity according to conventional methods. The extracted nucleic acid was divided into small portions and stored in a -80°C refrigerator.

[0031] 1.2 Design and synthesis of specific primers and probes

[0032] Using Primer 3 software, the fluorescent quantitative PCR forward primer and reverse primer were designed for the OC43 N protein gene sequence, and the probe was designed inside the gene. The design method is as follows: for the targeted target gene, TaqMan probes were designed using the sliding window method, the window...

Embodiment 2

[0047] Embodiment 2 fluorescent quantitative PCR method detects human coronavirus HKU1

[0048] 2.1 Extraction of nucleic acid

[0049] Take clinical samples from patients with acute respiratory infection, such as sputum, nasopharyngeal swabs, alveolar lavage fluid, etc. Nucleic acid was extracted according to a conventional method, and the obtained nucleic acid was dissolved in 50 μl of eluent. Quantification and purity testing of the extracted nucleic acids were carried out according to conventional methods. The extracted nucleic acid was divided into small portions and stored in a -80°C refrigerator.

[0050] 2.2 Design and synthesis of specific primers and probes

[0051] Using Primer 3 software, the fluorescent quantitative PCR forward primer and reverse primer were designed for the HKU1 N protein gene sequence, and probes were designed inside the gene. The method is as described in 1.2. Primers and probes were synthesized according to conventional methods, and ROX f...

Embodiment 3

[0065] Embodiment 3 fluorescent quantitative PCR method detects respiratory syncytial virus RSVB

[0066] 3.1 Extraction of nucleic acid

[0067] Take clinical samples such as sputum, nasopharyngeal swabs, alveolar lavage fluid, etc. from patients with acute respiratory infection, extract nucleic acid according to conventional methods, and dissolve the obtained nucleic acid in 50 μl eluent. Quantification and purity testing of the extracted nucleic acids were carried out according to conventional methods. The extracted nucleic acid was divided into small portions and stored in a -80°C refrigerator.

[0068] 3.2 Design and synthesis of specific primers and probes

[0069] Using Primer 3 software, the forward primer and reverse primer of fluorescent quantitative PCR were designed for the gene sequence of RSVB N protein, and the probe was designed inside the gene. The method is as described in 1.2. Primers and probes were synthesized according to conventional methods, and a C...

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Abstract

The invention provides primers and probes or a combination thereof of fluorescent quantitative PCR that can be used to detect diseases caused by human coronavirus and respiratory syncytial virus. In aspecific embodiment, the sequences of the primers and probes are as shown in SEQIDNOs: 1-3, and / or as shown in SEQIDNOs: 4-6, and / or as shown in SEQIDNOs: 7-9. The invention also provides applicationof the primers and probes or the combination thereof to preparation of reagents for detecting human coronavirus OC43, human coronavirus HKU1 and / or respiratory syncytial virus RSVB in samples.

Description

technical field [0001] The invention relates to the field of human respiratory virus detection, in particular to a fluorescent quantitative PCR method and application thereof which can be used in the field of detection of diseases caused by human coronavirus and respiratory syncytial virus. Background technique [0002] At present, infection is still a major threat to human health, especially acute respiratory infectious diseases, which have high morbidity and mortality due to their many types, rapid spread, and wide prevalence, posing a major threat to human health, social stability, and economic development. . The Global Burden of Diseases, Injuries and Risk Factors (GBD) study found that lower respiratory tract infections killed 650,000 children under 5 years of age and 1.08 million adults over 70 years of age in 2016, out of an estimated 2.38 million deaths worldwide in all age groups lower respiratory infection, making it the sixth leading cause of death in all age gro...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6851C12N15/11C12R1/93
CPCC12Q1/701C12Q1/6851C12Q2563/107C12Q2531/113
Inventor 王健伟任丽丽肖艳郭丽李建国
Owner INST OF PATHOGEN BIOLOGY CHINESE ACADEMY OF MEDICAL SCI
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