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Multi-purpose real-time fluorescent quantitative PCR kit, method and primer probe composition for detecting 2019 novel coronavirus

A real-time fluorescence quantitative and coronavirus technology, applied in the field of nucleic acid detection, can solve problems such as new coronavirus infection, severe acute respiratory syndrome, and dyspnea that cannot be ruled out, and achieve improved guiding significance, short detection cycle, and good repeatability Effect

Inactive Publication Date: 2021-04-30
NINGBO HEALTH GENE TECHNOLOGIES CO LTD +1
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Problems solved by technology

[0003] The pneumonia caused by human infection with the 2019 novel coronavirus (SARS-CoV-2) is called novel coronavirus pneumonia (COVID-19). Common signs include respiratory symptoms, fever, cough, shortness of breath and dyspnea. In severe cases, Infection can lead to pneumonia, severe acute respiratory syndrome, kidney failure and even death. So far, there is no specific treatment. Therefore, early detection and early isolation are the most effective measures to curb the development of the epidemic
The "Technical Guidelines for Laboratory Detection of Pneumonia Infected by Novel Coronavirus" issued by the National Health and Medical Commission clearly pointed out that the detection method of new coronavirus infection is reverse transcription real-time fluorescent quantitative PCR, and the detection method is mainly for the genome of the new coronavirus. Box 1ab (open readingflame 1ab, ORF1ab) and nucleocapsid protein (nucleocapsid protein, N), if the two target-specific reverse transcription real-time fluorescent quantitative PCR detection results of the new coronavirus in the same specimen are positive, the case is determined to be Positive, but at the same time, the guidelines also pointed out that due to the existence of negative cases caused by virus mutations, negative results cannot rule out new coronavirus infection

Method used

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  • Multi-purpose real-time fluorescent quantitative PCR kit, method and primer probe composition for detecting 2019 novel coronavirus
  • Multi-purpose real-time fluorescent quantitative PCR kit, method and primer probe composition for detecting 2019 novel coronavirus
  • Multi-purpose real-time fluorescent quantitative PCR kit, method and primer probe composition for detecting 2019 novel coronavirus

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] The presence of 2019-nCoV RNA in the respiratory and blood samples of suspected patients and non-suspected patients was detected by multiple reverse transcription real-time fluorescent quantitative PCR detection kits for 2019-nCoV respectively (respiratory tract samples of suspected patients are numbered A1, blood samples of suspected patients The number is A2, the number of respiratory samples from non-suspect patients is B1, and the number of blood samples from non-suspect patients is B2):

[0062] 1. Nucleic acid extraction: Use nucleic acid extraction reagents to automatically extract nucleic acids from samples A1, A2, B1, and B2 on an automatic nucleic acid extraction instrument at the same time, and store the extracted nucleic acid samples at -20°C.

[0063] 2. Prepare RT-PCR system: prepare RT-PCR reaction system according to 14 μL 2019 novel coronavirus detection master mix, 1 μL RT-PCR enzyme solution and 5 μL nucleic acid sample to be detected for each reaction...

Embodiment 2

[0067] Standard curve establishment and sensitivity test of multiple reverse transcription real-time fluorescent quantitative PCR detection kit for 2019 novel coronavirus:

[0068] 1. The positive recombinant plasmids comprising the S gene, ORF1ab gene, N gene and B2M gene after the concentration and purity were determined were respectively diluted 10 times to obtain from 1×10 2 ~1×10 8 A total of 7 dilutions of positive plasmids in copies / μL were used as standard templates. Prepare the reaction system according to Table 4, perform multiple reverse transcription real-time fluorescent quantitative PCR according to Table 5, obtain the fluorescence amplification curve and draw the standard curve.

[0069] 2. For the S gene ( Figure 17 ), at a concentration of 1×10 2 ~1×10 8 The amplification curve in the range of copies / μL presents a more typical S-type, the curves are evenly spaced, and have good correlation. The linear equation is y=-2.9351x+41.435, R2 =0.9981, the lowest ...

Embodiment 3

[0071] Multiple reverse transcription real-time fluorescent quantitative PCR detection kit specificity test for 2019 novel coronavirus:

[0072] 1. Using pathogenic microorganisms of common respiratory diseases (including human coronavirus 229E, human coronavirus NL63, human coronavirus OC43, human coronavirus HKU1, influenza A virus, parainfluenza virus, metapneumovirus, influenza B virus, respiratory tract Syncytial virus, rhinovirus, Boca virus, Chlamydia pneumoniae, Chlamydia trachomatis, Mycoplasma pneumoniae, and adenovirus) were used as templates to test the specificity of the kit.

[0073] 2. The test results are shown in Table 6. The results showed that the multiplex reverse transcription real-time fluorescent quantitative PCR detection kit for 2019 novel coronavirus was negative for pathogenic microorganisms of common respiratory diseases, which proved that the kit had good specificity.

[0074] Table 6 Kit Specificity Verification

[0075]

[0076]

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Abstract

The invention relates to a multi-purpose reverse transcription real-time fluorescent quantitative PCR detection kit for synchronously detecting a 2019 novel coronavirus ORF1ab gene, an N gene, an S gene and a human B2M gene as well as a special primer and probe combination thereof, and the kit adopts a single tube and four fluorescent channels to simultaneously detect three 2019 novel coronavirus genes and a human reference gene, and can detect the existence of the 2019 novel coronavirus RNA in respiratory tracts and blood samples. The kit has the advantages of short detection period, strong specificity, high sensitivity, low omission ratio and good repeatability, can perform quality monitoring on the extraction and amplification process of the sample according to the detection result of the human reference gene, and reduces the occurrence of false positive results caused by aerosol pollution through a dUTP-UNG enzyme anti-pollution system.

Description

[0001] This application claims the priority of the earlier application, the application number of which is CN202010170258.1, and the title of the invention is "a multiplex real-time fluorescent quantitative PCR kit, method and primer-probe composition for detecting 2019 novel coronavirus" . technical field [0002] The invention relates to the technical field of nucleic acid detection, in particular to a multiplex real-time fluorescent quantitative PCR kit, method and primer-probe composition for detecting 2019 novel coronavirus. Background technique [0003] The pneumonia caused by human infection with the 2019 novel coronavirus (SARS-CoV-2) is called novel coronavirus pneumonia (COVID-19). Common signs include respiratory symptoms, fever, cough, shortness of breath and dyspnea. In severe cases, Infection can lead to pneumonia, severe acute respiratory syndrome, kidney failure and even death. So far, there is no specific treatment. Therefore, early detection and early isola...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6851C12N15/11
CPCC12Q1/701C12Q1/6851C12Q2600/16C12Q2600/166C12Q2521/107C12Q2521/531C12Q2531/113C12Q2563/107C12Q2545/113C12Q2537/143Y02A50/30
Inventor 张迪骏曾县平张顺吴勇
Owner NINGBO HEALTH GENE TECHNOLOGIES CO LTD
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