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A digital pcr method for detecting group b1 adenovirus

An adenovirus and digital technology, applied in biochemical equipment and methods, recombinant DNA technology, microbial measurement/inspection, etc., can solve problems such as high incidence, affecting military work and training, etc.

Active Publication Date: 2022-03-08
THE FIFTH MEDICAL CENT OF CHINESE PLA GENERAL HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The incidence of mass respiratory adenovirus infection is high in the army, which affects the work and training of soldiers

Method used

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  • A digital pcr method for detecting group b1 adenovirus
  • A digital pcr method for detecting group b1 adenovirus
  • A digital pcr method for detecting group b1 adenovirus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0076] Embodiment 1, screen PCR site and design primer and probe

[0077] The research team compared the H genes of group B1 adenoviruses and found that the specificity of the H genes of group B1 adenoviruses was high, and then selected different positions on the H genes as targets to design three sets of primer probes. The sequence is shown in Table 1:

[0078] Table 1. Primer and probe sequences for detection of group B1 adenoviruses

[0079]

[0080] Using adenovirus type 3 as a template, use the above three sets of primers for qPCR detection, and use takara's OneStep PrimeScript for detection TM RT-PCR Kit (Perfect Real Time) (Code No.RR064) kit, total reaction system 25μl, of which 2X one Step RT-PCR Buffer 12.5μl, TaKaRa Ex TaqHs 0.5μl, RoxReference DyeⅡ0.5μl, primer-probe mixture 3 μl, RNase Free dH 2 O 6.5 μl, DNA 2 μl.

[0081] The reaction conditions were 95°C for 10s, 95°C for 5s, 60°C for 34s, 40 cycles (collecting fluorescent signals). The experiment was...

Embodiment 2

[0094] Embodiment 2, screening internal reference gene and primer probe thereof

[0095] The research group used the respiratory cell housekeeping gene RPP30 as an internal reference gene, and set up 3 sets of primer controls, as shown in Table 2:

[0096] Table 2. Primer and probe sequences for detecting RPP30 internal reference genes

[0097]

[0098] Throat swab samples of patients kept by the research group were selected for digital PCR detection. The instrument used TARGETING ONE system including droplet generator and droplet reader (Xinyi Biology) A300 Fast Thermal Cycler rapid gradient PCR instrument (LongGene Langji) .

[0099] Reaction system: master mix 15 μl [primer-probe mixture: adv7 primer 500nM, probe 200nM, RPP30 primer 600nM, probe 300nM (concentration is the final concentration of the PCR system)], 3μl ADV7+3μl RPP30, 7μl H 2O. 2 μl template.

[0100] The amplification conditions were: 95°C for 600s, 94°C for 30s, 55°C for 60s, 42 cycles, 12°C for 300...

Embodiment 3

[0106] Embodiment 3, B1 primer probe detects the result of AdV3, AdV7, AdV11, AdV14, AdV55 type adenovirus respectively

[0107] The detection effect of the B1 group adenovirus detection primer probe screened in Example 1 and the internal reference gene primer probe screened in Example 2 in digital PCR detection was verified. Among them, the throat swab extracts from healthy people mainly provide internal reference genes for the samples to be tested.

[0108] Instruments: Xinyi TD-1 droplet digital PCR system, A300 Fast Thermal Cycler (LongGene).

[0109] Reaction system: master mix 15 μl [primer-probe mixture: adv7 primer 500nM, probe 200nM, RPP30 primer 600nM, probe 300nM (concentration is the final concentration of the PCR system)], 3μl ADV7+3μl RPP30, 7μl H 2 O. 2 μl template.

[0110] Amplification conditions: 95°C for 600s, 94°C for 30s, 55°C for 60s, 42 cycles, 12°C for 300s Note: The temperature change speed is set to 1.5°C / s. The result is as follows:

[0111] T...

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Abstract

The invention provides a primer for detecting B1 group adenovirus, and a probe-primer combination, a reagent and a kit including the primer. In the present invention, the aforementioned product detection method is highly accurate, has higher sensitivity than general PCR, and can be quantitatively detected.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a digital PCR method for detecting B1 group adenovirus. Background technique [0002] Currently, digital PCR includes droplet PCR detection method and chip detection method. In the chip-based detection method, the number of effective reaction chambers on a single chip is generally only a few thousand, which is far less than that of the droplet-based detection method. Therefore, the dynamic range of chip-based digital PCR is narrower than that of droplet-based PCR. The droplet PCR detection method disperses the sample into water-in-oil reaction units, and then performs real-time or endpoint fluorescence analysis on each reaction unit. [0003] Digital PCR (digitalPCR, dPCR) is a newly developed third-generation PCR technology, mainly including large-scale integrated microfluidic chip digital PCR (chamber based digital PCR, cdPCR) and droplet digital PCR (droplet digital PCR, ddPCR)....

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/686C12N15/11
Inventor 陈威巍许文杨俊连杨欣欣徐哲涂波黄磊
Owner THE FIFTH MEDICAL CENT OF CHINESE PLA GENERAL HOSPITAL
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