Reverse transcription fluorogenic quantitative PCR primer for rapidly identifying high virulent Newcastle disease virus (NDV) strain and identification method

A fluorescence quantitative, virulent strain technology, applied in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc., can solve problems such as high cost and poor sensitivity, and achieve simple and specific detection methods. , the effect of high detection sensitivity

Inactive Publication Date: 2016-04-27
INST OF ANIMAL SCI & VETERINARY MEDICINE SHANDONG ACADEMY OF AGRI SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In order to solve the above problems of high cost and poor sensitivity in the identification of strong and weak virulent Newcastle disease virus, the present invention provides a fast, sensitive and low-cost method for distinguishing strong and weak NDV strains

Method used

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  • Reverse transcription fluorogenic quantitative PCR primer for rapidly identifying high virulent Newcastle disease virus (NDV) strain and identification method
  • Reverse transcription fluorogenic quantitative PCR primer for rapidly identifying high virulent Newcastle disease virus (NDV) strain and identification method
  • Reverse transcription fluorogenic quantitative PCR primer for rapidly identifying high virulent Newcastle disease virus (NDV) strain and identification method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Embodiment 1: Optimization of RT-qPCR reaction conditions

[0034] The determination of the reaction conditions is mainly to optimize the primer concentration and annealing temperature. The reaction system is 20 μL, including 10 μL of 2×OneStepSYBRRT-PCRBuffer; 0.4 μL of ExTaqHS; 0.4 μL of PrimescriptrtenzymemixⅡ; 1 μL of NDVFrealtime-1 and NDVFrealtime-2 primers (5-100 μM); RnaseFreedH 2 O5.2μL; TotalRNA2μL. The reaction conditions were: reverse transcription at 42°C for 5 minutes; pre-denaturation at 95°C for 3 minutes; 40 cycles of amplification at 94°C for 10s, 51-57°C for 10s, and 72°C for 20s, and fluorescence signals were collected for each cycle. The primer concentration was 10 μM, 20 μM, 30 μM, 40 μM, and 50 μM in 5 concentration gradients, and the annealing temperature was 51 °C, 53 °C, 54 °C, 55 °C, and 57 °C at 5 reaction temperatures, and each reaction condition was repeated three times. . The results were judged according to the amplification curve and ...

Embodiment 2

[0035] Embodiment 2: establish standard curve

[0036] According to the determined optimal reaction conditions, with 10 8 、10 7 、10 6 、10 5 、10 4 、10 3 Copy / μL RNA standards with 6 concentration gradients were used as templates for the establishment of RT-qPCR standard curves. The reaction system of each sample is the same, 20 μL, including 10 μL of 2×OneStepSYBRRT-PCRBuffer; 0.4 μL of ExTaqHS; 0.4 μL of PrimescriptrtenzymemixⅡ; 2 O5.2μL; TotalRNA2μL.

[0037] The reaction conditions were determined as follows: reverse transcription at 42°C for 5 minutes; pre-denaturation at 95°C for 3 minutes; 40 cycles of amplification at 94°C for 10s, 53°C for 10s, and 72°C for 20s. repeated to obtain the respective kinetic curves, such as image 3 . Then obtain the standard curve, the linear equation of the standard curve is Y=-3.307X+2.657, such as Figure 4 . The results showed that the repeatability of each dilution was good, and the established standard curve was qualified. ...

Embodiment 3

[0038] Embodiment 3: sensitivity test

[0039] respectively by 10 6 、10 5 、10 4 、10 3 、10 2 、10 1 、10 0 The copy / μL standard product is used as a template, and other conditions are the same as in Example 2, and RT-qPCR amplification is performed, and the detection limit value of the copy number detection method of the lowest dilution that can be effectively amplified. The results showed that the minimum concentration of the template that could be detected by fluorescent quantitative PCR was 10 0 copy / μL( Figure 5 ), which indicated that the detection limit of RT-qPCR was 1 copy / μL.

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Abstract

The invention relates to the virus detection technologies in the fields of molecular biology and molecular virology, and in particular relates to a reverse transcription fluorogenic quantitative PCR primer for rapidly identifying a high virulent Newcastle disease virus (NDV) strain and a method for identifying the high virulent NDV strain by adopting the PCR primer. The method comprises the following steps: extracting RNA of a to-be-identified virulent strain, carrying out RT-qPCR amplification, and identifying that the to-be-identified virus is the high virulent NDV strain when viral nucleic acid is successfully amplified; and identifying that the to-be-identified virus is not the high virulent NDV strain when the viral nucleic acid is not successfully amplified. According to the identification method, only once RT-qPCR amplification is needed, whether the to-be-identified virus is the high virulent NDV strain or not can be judged, and the detection method is simple, convenient and flexible, and is easy to operate; the detected minimum concentration of RNA is 1 copy/mu L, and the detection sensitivity is high; the design primer only can amplify the high virulent NDV strain, and can not amplify low virulent strains and other virulent strains, and therefore, the specificity is strong.

Description

technical field [0001] The invention relates to virus detection technology in the fields of molecular biology and molecular virology, in particular to a reverse transcription fluorescence quantitative PCR primer for rapidly identifying virulent Newcastle disease strains, and also relates to an identification method using the primer. Background technique [0002] Newcastle disease (Newcastledisease, ND) is an acute, highly contagious poultry infectious disease caused by Newcastle disease virus (Newcastlediseasevirus). Huge economic loss. NDV, also known as avian paramyxovirus type 1, belongs to the genus Avian Mumps virus in the order Single-strand Negavirales, Paramyxoviridae, and Paramyxoviridae in classification. The NDV genome contains 6 open reading frames, which encode six structural proteins L-HN-F-M-P-NP sequentially from 5' to 3', among which F protein is one of the most important glycoproteins, the strength of virus virulence and its cleavage site The amino ac...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
CPCC12Q1/701C12Q1/6851C12Q2563/107C12Q2545/114C12Q2521/107
Inventor 张琳胡北侠刘伟杰张秀美杨少华黄艳艳许传田黄庆华
Owner INST OF ANIMAL SCI & VETERINARY MEDICINE SHANDONG ACADEMY OF AGRI SCI
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