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Multi-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 dyspnea, death, pneumonia, etc., and achieve the effects of short detection cycle, high sensitivity, and low missed detection rate

Inactive Publication Date: 2020-06-12
NINGBO HEALTH GENE TECHNOLOGIES CO LTD +1
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AI Technical Summary

Benefits of technology

This patented technology allows researchers to quickly diagnose new viruses that may cause serious illness or even lead them into extinction without causing any harm during their first stages of spreading out across different regions worldwide.

Problems solved by technology

This patented technical problem addressed in this patents relates to identifying patients infected or suspected from COVID19 who have been previously identified through testing methods like qPCR. These tests may result in false positives indicating potential future viral transmissions.

Method used

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

[0053] 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):

[0054] 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.

[0055] 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

[0059] 2019 Novel Coronavirus Multiplex Reverse Transcription Real-Time Fluorescent Quantitative PCR Detection Kit Standard Curve Establishment and Sensitivity Test:

[0060] 1. The positive recombinant plasmids containing S gene, ORF1ab gene, N gene and B2M gene were respectively diluted 10 times after the concentration and purity were determined, and obtained 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.

[0061] 2. For the S gene ( Figure 17 ), at a concentration of 1×10 2 ~1×10 8 The amplification curve within the range of copies / μL presents a typical S-shape, the curves are evenly spaced, and have good correlation. The linear equation is y=-2.9351x+41.435, R 2 =0.9981, the lowest de...

Embodiment 3

[0063] Multiple reverse transcription real-time fluorescent quantitative PCR detection kit for 2019 novel coronavirus Specificity test:

[0064] 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 Syncytial virus, rhinovirus, boca virus, Chlamydia pneumoniae, Chlamydia trachomatis, Mycoplasma pneumoniae, and adenovirus) were used as templates to test the specificity of the kit.

[0065] 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.

[0066] Table 6 Kit specificity verification

[0067]

[0068]

[0069] The pres...

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Abstract

The invention relates to a multiple reverse transcription real-time fluorescent quantitative PCR detection kit for synchronously detecting an ORF1ab gene, an N gene, and an S gene of 2019 novel coronavirus and a human B2M gene, and a special primer and probe combination thereof. According to the kit, three 2019 novel coronavirus genes and a human reference gene are simultaneously detected by adopting a single-tube four-fluorescence channel, and existence of 2019 novel coronavirus RNA in respiratory tracts and blood samples can be detected. The kit is short in detection period, high in specificity, high in sensitivity, low in omission ratio and good in repeatability, quality monitoring can be performed on an extraction and amplification process of the sample through a detection result of the human reference gene, and a false positive result caused by aerosol pollution is reduced through a dUTP-UNG enzyme anti-pollution system.

Description

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Claims

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Application Information

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Owner NINGBO HEALTH GENE TECHNOLOGIES CO LTD
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