Primers, probe and kit for detecting duck-origin coronavirus through fluorescence quantitative RT-PCR (Reverse Transcription-Polymerase Chain Reaction)

A technology of RT-PCR and coronavirus, which is applied in the field of molecular biology detection of viruses, can solve problems such as the lack of new molecular biology detection methods, achieve important economic value and social significance, strong specificity, and avoid false positive effects

Inactive Publication Date: 2011-09-14
HENAN INST OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is still a lack of effective new molecular biological detection methods for this virus

Method used

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  • Primers, probe and kit for detecting duck-origin coronavirus through fluorescence quantitative RT-PCR (Reverse Transcription-Polymerase Chain Reaction)
  • Primers, probe and kit for detecting duck-origin coronavirus through fluorescence quantitative RT-PCR (Reverse Transcription-Polymerase Chain Reaction)
  • Primers, probe and kit for detecting duck-origin coronavirus through fluorescence quantitative RT-PCR (Reverse Transcription-Polymerase Chain Reaction)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1: Establishment of Fluorescent Quantitative RT-PCR Detection Method

[0029] (1) Processing of samples to be tested: take 1.0 g of corresponding tissues and organs (such as trachea, heart, kidney, etc.), put them into a 1.5 mL sterilized centrifuge tube, cut them into pieces, and add PBS buffer solution sterilized by autoclaving at a ratio of 1 g. : 5mL, repeatedly freeze-thawed at -20℃ for 3 times, centrifuged at 1000rpm for 5min; discarded the precipitate, took the supernatant in another 1.5mL sterilized centrifuge tube, centrifuged at 8000rpm for 10min; discarded the precipitate, took the supernatant in another 1.5mL Centrifuge at 12,000 rpm for 10 min in a 1 mL sterilized centrifuge tube; discard the precipitate, take the supernatant into another 1.5 mL sterilized centrifuge tube, and filter with a 0.22 μm filter.

[0030] (2) Handling of standard viruses

[0031] The duck-derived coronavirus ZZ2004 strain was inoculated into 10-day-old SPF chicken embryos...

Embodiment 2

[0074] Embodiment 2: Fluorescence quantitative PCR detects the distribution of virus in chicken body

[0075] After using the method and kit established in the above-mentioned Example 1 to artificially infect the duck-derived coronavirus ZZ2004 strain in 7-day-old SPF chicks, the content of the virus in each organ was detected and found that multiple organs of the chicken were infected with the virus. After infection Viral nucleic acid can be detected in lungs, kidneys, glandular stomach, duodenum, cecum and other organs within 3 hours; viral nucleic acid can be detected in liver, trachea, spleen, and ileum 6 hours after infection; Viral nucleic acid was detected in the bursa, rectum, and thymus, and the duration of viral nucleic acid in each tissue was: duodenum (957h), cecum (957h), liver (954h), rectum (951h), glandular stomach (717h ), bursa (711h), pancreas (696h), kidney (597h), thymus (591h), jejunum (576h), trachea (498h), lung (405h), heart (384h), ileum (258h), splee...

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Abstract

The invention relates to primers, probe and kit for detecting duck-origin coronavirus through fluorescence quantitative RT-PCR (Reverse Transcription-Polymerase Chain Reaction). Nucleotide sequences of an upper primer, a lower primer and the probe are 5'-AGGTAGTGGTGTTCCTGATAATGAG-3', 5'-ACGCATCTGGGACTGGTTTTC-3' and 5'-CCTGGCTTATACCTGGCTTGGCGTCT-3'; and the kit comprising the primers and the probefor detecting the duck-origin coronavirus through the fluorescence quantitative RT-PCR is established. The kit has the advantages of high specificity, high flexibility, accurate quantification, easiness and convenience in operation, short time, high efficiency and low cost and can be used for detecting a large number of samples and providing basis for treatment, prevention and control of IB (Infectious Bronchitis) in a short time; and compared with the conventional PCR detection, the primers, the probe and the kit can be used for effectively preventing occurrence of false positive, and has decreased detectable amount of virus and higher sensitivity.

Description

technical field [0001] The invention relates to the field of molecular biology detection of viruses, in particular to a primer, a probe and a kit for detecting duck-derived coronavirus by fluorescent quantitative RT-PCR. Background technique [0002] Fluorescent quantitative PCR technology was first introduced by Applied Biosystems in 1996. It is an emerging molecular biology detection method integrating PCR amplification technology, probe hybridization technology and fluorescence resonance energy transfer spectroscopy technology. It realizes PCR from qualitative to Quantitative leap. The principle of fluorescent quantitative PCR technology is: add a fluorescent group to the PCR reaction system, use the accumulation of fluorescent signals to monitor the entire PCR process in real time, and finally use the standard curve to quantitatively analyze the unknown template. This method is currently one of the most advanced detection methods used in virus detection in the world. Co...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68G01N21/64C12R1/93
Inventor 刘兴友刘明成王丽荣胡建和
Owner HENAN INST OF SCI & TECH
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