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RT-LAMP (Reverse Transcription Loop-Mediated Isothermal Amplification) primer group and kit for detecting avian influenza viruses and applications of RT-LAMP primer group and kit

A technology of RT-LAMP and avian influenza virus, applied in the direction of microorganism-based methods, DNA/RNA fragments, microorganisms, etc., can solve the problems of inapplicability to rapid detection at the grassroots level or on-site, the need for expensive equipment, and the long detection cycle, etc., to achieve Wide range of objects, simple equipment and operation, and strong applicability

Active Publication Date: 2015-05-27
河北伯瑞动物药业有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These methods have played an important role in virus detection, but they generally have disadvantages such as cumbersome operation, long detection cycle, and need for expensive instruments and equipment, and are not suitable for rapid detection at the grassroots level or on-site

Method used

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  • RT-LAMP (Reverse Transcription Loop-Mediated Isothermal Amplification) primer group and kit for detecting avian influenza viruses and applications of RT-LAMP primer group and kit
  • RT-LAMP (Reverse Transcription Loop-Mediated Isothermal Amplification) primer group and kit for detecting avian influenza viruses and applications of RT-LAMP primer group and kit
  • RT-LAMP (Reverse Transcription Loop-Mediated Isothermal Amplification) primer group and kit for detecting avian influenza viruses and applications of RT-LAMP primer group and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1 The best embodiment of the kit for detecting avian influenza virus by RT-LAMP method

[0045] The kit of this embodiment includes the following RT-LAMP primer sets, see Table 1. The kits are divided into A box and B box, and A box is an RNA extraction kit. The composition and storage conditions of the kit are shown in Table 2. Box B is an avian influenza virus reverse transcription loop-mediated isothermal amplification (RT-LAMP) kit. The kit composition and storage conditions are shown in Table 3.

[0046] Among them, the RT-LAMP reaction solution consists of 50uL 10 × Bst DNA Buffer, 50uL MgCl2 solution with a concentration of 25mM, 60uL dNTPs solution with a concentration of 2.5mM, and 120uL nuclease-free water.

[0047] The reverse transcriptase consisted of 20 uL of AMV reverse transcriptase at a concentration of 200 U / μL and 10 uL of an RNase inhibitor at a concentration of 50 U / μL.

[0048] The fluorescent chromogenic reagent contains calcein and MnCl...

Embodiment 2

[0057] Example 2 Non-diagnostic purpose using RT-LAMP method to detect the detection method of avian influenza virus

[0058] The detection method of this embodiment uses the kit of Embodiment 1 for detection. The detection method of this embodiment comprises the following steps:

[0059] 1. Viral RNA extraction Avian influenza virus RNA was extracted by one-step method of guanidinium isothiocyanate-phenol-chloroform. The specific steps are as follows: (1) Denaturation. Add 750 μL TRIzol LS Regent to 250 μL of the sample to be tested, the positive control and the negative control, shake vigorously for 2 min, and place at room temperature for 10 min. Add 250 μL of chloroform, shake vigorously for 15 s, leave at room temperature for 10 min, centrifuge at 12,000 g for 10 min at 4°C, and transfer the upper aqueous phase (about 500–600 μL) into a new centrifuge tube. Among them, the samples to be tested can be intestinal contents, lungs, air sacs, intestines, spleen, liver, he...

Embodiment 3

[0069] The sensitivity test of the RT-LAMP of embodiment 3 avian influenza virus

[0070] Measured by an ultra-micro spectrophotometer, a nucleic acid template with a total RNA concentration of 40 ng / μL of the detected avian influenza virus was taken for determination. At the same time, RT-LAMP and RT-PCR methods were used to detect the 10-fold diluted avian influenza virus nucleic acid template.

[0071] The RT-LAMP reaction operation process of avian influenza virus is the same as embodiment 2, and the RT-PCR reaction is composed as follows:

[0072] The product cDNA obtained by reverse transcription of the avian influenza virus nucleic acid was identified by RT-PCR. In the 25μL system, add 2× Taq Mix, cDNA, upstream primer F (10 uM), downstream primer R (10 uM) and dd H 2 O. Place in PCR machine reaction, pre-denaturation at 94°C for 1min; pre-denaturation at 94°C for 30s, annealing at 56°C for 30s, extension at 72°C for 15s, a total of 30 cycles; extension at 72°C ...

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Abstract

The invention discloses an RT-LAMP (Reverse Transcription Loop-Mediated Isothermal Amplification) primer group and a kit for detecting avian influenza viruses and applications of the RT-LAMP primer group and the kit. The primer group consists of a pair of outer primers and a pair of inner primers, wherein the sequences of the two outer primers are shown in SEQ ID No.1 and SEQ ID No.2, the sequences of the two inner primers are shown in SEQ ID No.3 and SEQ ID No.4, and the primers can be used for detecting the avian influenza viruses effectively. The kit comprises the primers and can be used for detecting the avian influenza viruses. The invention also provides a method for detecting the avian influenza viruses by adopting the kit. The detection result of the kit can be used for intuitively performing judgment quickly and accurately according to color changes of a reaction solution. The RT-LAMP primer group and the kit disclosed by the invention have the characteristics of strong specificity, high sensitivity, fastness and accurate avian influenza viruse detection, and are particularly suitable for field and basement layer detection.

Description

technical field [0001] The invention relates to the technical field of bird flu virus detection. Background technique [0002] Avian Influenza (AI), also known as "true chicken plague" or "European chicken plague", is an infectious disease of poultry caused by avian influenza virus (AIV). It mainly occurs in chickens, ducks, geese, pigeons, etc., and people can also be infected. According to its clinical manifestations, it can be divided into highly pathogenic, low pathogenic and non-pathogenic avian influenza. Among them, highly pathogenic avian influenza is listed as a class A animal disease by the World Organization for Animal Health because of its rapid spread and great harm, and it is listed as a class I animal disease in my country. AIV detection methods mainly include: virus isolation, RT-PCR, real-time fluorescence quantitative PCR. These methods have played an important role in virus detection, but they generally have disadvantages such as cumbersome operation, l...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
CPCC12Q1/6844C12Q1/70
Inventor 袁万哲刘聚祥孙继国陈立功刘静王庚南
Owner 河北伯瑞动物药业有限公司
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