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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 and RT-PCR technology, applied in the field of biomedical detection, can solve the problems of low sensitivity, high experimental cost, and long time consumption, and achieve the effects of reducing economic losses, saving time, and reducing detection costs

Inactive Publication Date: 2011-10-12
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 cows, add sterilized normal saline to prepare a 1:5 suspension, centrifuge at 4000 r / min for 10 min, 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 into a 1.5 mL centrifuge tube, add 1 m TRIZOL, mix well, and leave at room temperature for 10 min.

[0033] (2) Add chloroform at 200 μL chloroform / mL Trizol, shake vigorously and mix, and place at room temperature for 15 minutes (note: the vortex shaker is disabled to avoid genomic DNA fragmentation); centrifuge at 12,000g for 15 minutes at 4°C. Aspirate the upper aqueous phase to 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 in a 4°C refrigerator, if only extract RNA, discard the lower phenolic phase).

[0034] (3) Add isopropanol t...

Embodiment example 2

[0050] Example 2: Optimization of reaction conditions of RT-PCR kit for bovine parainfluenza virus type 3

[0051] The specific upstream and downstream primers P1 and P2 of bovine parainfluenza virus type 3 were tested respectively, and the annealing temperature and primer concentration were selected to optimize the conditions of the kit. The annealing temperature of PCR starts at 54°C, 54-62.0°C is 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 primer concentration is 0.2 Within the range of ~1.0μM, they were 1 μmol / L, 0.8 μmol / L, 0.6 μmol / L, 0.5 μmol / L, and 0.4 μmol / L, respectively. The PCR amplification effect was observed by gradually increasing the primer concentration. Other reaction conditions were unchanged.

[0052] result:

[0053] Figure 4 : M.DL2000 ladder from left to right; annealing temperatures from 1 to 10 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, respectively. The picture show...

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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 biology detection method. Specifically, it relates to a RT-PCR detection method for rapid diagnosis of bovine parainfluenza virus type 3. It also relates to the application of 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) infection, which can be divided into calf type and adult cattle type. Calf-type pneumonia, also known as calf-endemic pneumonia, is a contagious contagious disease that affects calves from 2 weeks to several months of age and is characterized by fever, dyspnea, serosal, mucoid or purulent rhinorrhea, and cough. Because the disease mostly occurs in cattle after transportation, the virus is also called transportation fever virus. Bovine parainfluenza virus type 3 is a member of the Paramyxoviridae family of Paramyxo...

Claims

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Application Information

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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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