Primer, probe series and method for multiple real time fluorescent RT-PCR detection of H5, H7 and H9 subtype bird flu

A RT-PCR, real-time fluorescence technology, applied in biochemical equipment and methods, microbial determination/inspection, etc., can solve problems such as high variability, and achieve good specificity, simple operation, accurate and sensitive results.

Inactive Publication Date: 2005-09-21
TAITAI GENOMICS +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

This new method uses powerful techniques like polymerase chain reaction (PC) for DNA analysis with fast response times compared to traditional methods such as gel electrophoretic or immunological assays. It achieves this through efficient amplifying and detecting gene segments from samples containing both viruses A/B/C). By doing these operations at once, there are more precise data than current technologies but less expensive because they only require multiple steps instead of just two.

Problems solved by technology

The technical problem addressed in this patents relates to developing a new way to quickly identify and accurately monitor major human respiratory illness threatening agents such as swine fever coronaria vims, severe hemorrhages associated with porcinera fowls, salmon sialitis virus, Newcastle Diseas To address these issues, the inventors developed several novel techniques called multiplex real -world fluorescein qistype immunology assays (MFA). These technologies aim to provide better tools for early diagnosis and prevention efforts against pandemics like Spanishfluand Novel Bird Arteries.

Method used

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  • Primer, probe series and method for multiple real time fluorescent RT-PCR detection of H5, H7 and H9 subtype bird flu
  • Primer, probe series and method for multiple real time fluorescent RT-PCR detection of H5, H7 and H9 subtype bird flu
  • Primer, probe series and method for multiple real time fluorescent RT-PCR detection of H5, H7 and H9 subtype bird flu

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] (1) Preparation of the template to be tested

[0049] Method 1 Use the QIAamp Viral RNA Mini kit to extract the genomic RNA of avian influenza virus in samples from various sources. The specific operation steps are as follows:

[0050] a. Take 560 μl of AVL buffer containing carrier RNA to a 1.5ml microcentrifuge tube;

[0051] b. Add 140 μl of plasma, serum, urine sample, cell culture supernatant or PBS cleaning solution of swab, and vortex for 15 sec;

[0052] c. Incubate at room temperature (15-25°C) for 10 minutes;

[0053] d. Centrifuge briefly so that the liquid on the cover is centrifuged into the centrifuge tube;

[0054] e. Add 560 μl of absolute ethanol, vortex for 15 sec, centrifuge briefly, and centrifuge the liquid on the cover into the centrifuge tube;

[0055] f. Carefully pipette 630 μl of the above liquid into the QIAamp spin column (placed in a 2 ml collection tube), without wetting the edge of the tube, and centrifuge at 8000 rpm for 1 min. Discar...

Embodiment 2

[0071] Duplex real-time fluorescent RT-PCR detection of avian influenza H5 and H9 subtypes

[0072] In a 50 μl reaction system, use specific primers and probes for avian influenza H5 and H9 subtypes to perform double real-time fluorescent RT-PCR to detect positive samples containing avian influenza virus H5 and H9 subtype genomic RNA, and the results can be obtained Specific fluorescence curve ( image 3 ).

Embodiment 3

[0074] Duplex real-time fluorescent RT-PCR detection of avian influenza H7 and H9 subtypes

[0075] In a 50 μl reaction system, use specific primers and probes for avian influenza H7 and H9 subtypes to perform double real-time fluorescent RT-PCR to detect positive samples containing avian influenza virus H7 and H9 subtype genomic RNA, and the results can be obtained Specific fluorescence curve ( Figure 4 ).

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Abstract

This invention discloses an nucleotide priemr tested by multiple real-time fluorescence RT-PCR, probe sequence and its method suitable for high-pathogenicity fowl-influenza H5, H7, H9 hypotype, relating to the nucleotide fragment RT-PCR augmenting primer and its complementary sequence, and the parting testing method of fowl-influenza H5, H7, H9 hypotype .

Description

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Claims

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Application Information

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Owner TAITAI GENOMICS
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