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Reverse transcription-polymerase chain reaction (RT-PCR) detection method and kit for quickly diagnosing bovine parainfluenza virus 3 (BPIV-3)

A bovine parainfluenza virus, RT-PCR technology, applied in the field of biomedical detection, can solve the problems of high experimental cost, low sensitivity, long time consumption, etc., to save time, reduce economic losses, and reduce detection costs.

Inactive Publication Date: 2013-02-27
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This detection method has problems such as low sensitivity and long time-consuming, and some samples have anti-complement activity, which affects the detection effect
The cost of animal experiments is high, and consumes a lot of manpower and material resources, and the economic benefit is low

Method used

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  • Reverse transcription-polymerase chain reaction (RT-PCR) detection method and kit for quickly diagnosing bovine parainfluenza virus 3 (BPIV-3)
  • Reverse transcription-polymerase chain reaction (RT-PCR) detection method and kit for quickly diagnosing bovine parainfluenza virus 3 (BPIV-3)
  • Reverse transcription-polymerase chain reaction (RT-PCR) detection method and kit for quickly diagnosing bovine parainfluenza virus 3 (BPIV-3)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment example 1

[0029] 1 Sample collection and processing

[0030] Collect nasal swab or throat swab samples from sick cattle, add sterilized saline to the above samples to prepare a 1:5 suspension, centrifuge at 4000r / min for 10min, and take the supernatant for later use.

[0031] 2 Extraction of sample RNA template

[0032] (1) Take 330 μL of the supernatant from step 1, add it to a 1.5 mL centrifuge tube, add 1 m TRIZOL, mix well, and place at room temperature for 10 min.

[0033] (2) Add chloroform at 200 μL chloroform / mL Trizol, vortex vigorously and mix well, then place at room temperature for 15 minutes (Note: Vortex shaker is disabled to avoid genomic DNA fragmentation); centrifuge at 12,000 g for 15 minutes at 4°C. Aspirate the upper aqueous phase into another centrifuge tube (Note: Do not aspirate the middle interface; if DNA and protein are extracted at the same time, keep the lower phenolic phase and store in a 4°C refrigerator; if only RNA is extracted, discard the lower phenoli...

Embodiment example 2

[0050] Implementation Case 2: Optimization of Reaction Conditions of RT-PCR Kit for Bovine Parainfluenza Virus Type 3

[0051] The bovine parainfluenza virus type 3 specific upstream and downstream primers P1 and P2 were tested respectively, and the conditions of the kit were optimized by selecting annealing temperature and primer concentration that had a great influence on them. The annealing temperature of PCR starts at 54°C, and 54-62.0°C are 56.5°C, 57.3°C, 58.1°C, 58.9°C, 59.7°C, 60.5°C, 61.3°C, 62.1°C, 62.9°C, 63.7°C; The range of ~1.0μM is 1μmol / L, 0.8μmol / L, 0.6μmol / L, 0.5μmol / L, 0.4μmol / L, respectively, by gradually increasing the primer concentration, observe the effect of PCR amplification. Other reaction conditions remained unchanged.

[0052] result:

[0053] Figure 4 : M.DL2000 ladder from left to right; 1~10 annealing temperatures are 56.5°C, 57.3°C, 58.1°C, 58.9°C, 59.7°C, 60.5°C, 61.3°C, 62.1°C, 62.9°C, 63.7°C. The picture shows that the optimal annealin...

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Abstract

The invention discloses a reverse transcription-polymerase chain reaction (RT-PCR) detection method and a kit for quickly diagnosing bovine parainfluenza virus 3 (BPIV-3), and belongs to the field of biomedical detection. The invention adopts the technical scheme that the method comprises acquisition of a sample, extraction of virus RNA, design optimization of a specific primer, RT-PCR amplification, agarose gel electrophoresis and result judgment; the specific primer is designed for a specific fragment of BPIV-3 nucleoprotein (NP) genes, and the upstream and downstream sequences of the specific primer are respectively P1: 5'-GGATGTTTGGGAGTGATCTTGAGTA-3' and P2: 5'-TGTGTTGAAAAATGAAGCAAGACCT-3'; the quick RT-PCR diagnosing kit for detecting the BPIV-3 comprises a sample virus RNA extracting reagent, a reverse transcription reagent, a PCR reagent, a positive reference substance and a negative reference substance; and by verifying, the method is sensitive and specific and has applicationprospect.

Description

technical field [0001] The invention belongs to the field of biomedical detection, and relates to a microbial molecular biological detection method. Specifically relates to a RT-PCR detection method for rapid diagnosis of bovine parainfluenza virus type 3. It also relates to the application of the RT-PCR kit for rapid diagnosis of bovine parainfluenza virus type 3. Background technique [0002] Bovine parainfluenza is caused by Bovine parainfluenza virus 3 (BPIV-3), which can be divided into calf type and adult bovine type. Calf type, also known as calf enzootic pneumonia, is a contagious disease affecting calves aged 2 weeks to several months, characterized by fever, dyspnea, serous fluid, mucous or purulent rhinorrhea and cough. Because the disease mostly occurs in transported cattle, the virus is also known as transport fever virus. Bovine parainfluenza virus type 3 is a member of the genus Paramyxovirus in the family Paramyxoviridae, and is a single-stranded negative ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/68C12R1/93
Inventor 郭爱珍刘晓乐张敏敏陈颖玉胡长敏陈焕春
Owner HUAZHONG AGRI UNIV
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