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Rabies virus detecting fluorescence quantitative PCR kit and application thereof

A fluorescent quantitative and rabies virus technology, applied in the direction of fluorescence/phosphorescence, microbe-based methods, microbiological measurement/inspection, etc., can solve the problem of not being able to calculate the starting DNA or RNA copy number

Active Publication Date: 2009-10-28
WUHAN SANLI BIO TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Since there is no linear relationship between the amount of final PCR product and the amount of starting template, the starting DNA or RNA copy number cannot be calculated based on the amount of final PCR product

Method used

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  • Rabies virus detecting fluorescence quantitative PCR kit and application thereof
  • Rabies virus detecting fluorescence quantitative PCR kit and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] The fluorescent quantitative PCR kit composition of embodiment 1 rabies virus and reaction condition thereof

[0030] A). Kit composition:

[0031] a) The composition of the kit is as follows: (10 reactions)

[0032] RNA extraction solution A (4ml / tube) RNA extraction solution B (800μl / tube)

[0033] Reverse transcription reaction solution (90μl / tube) Reverse transcriptase (10μl / tube)

[0034] RNase inhibitor (400U / tube)

[0035] Strong Positive Standard (100-fold diluted standard 20μl / tube, four tubes in total)

[0036] PCR reaction tube (sterile, RNase and DNase free) DEPC H 2 O (2ml / tube)

[0037] Negative standard (50μl / tube) Borderline positive standard (50μl / tube)

[0038] Fluorescent quantitative PCR reaction solution (125μl / tube)

[0039] (RNA extraction solution A is a product of Invitrogen Company. b) reverse transcription reaction solution, d) reverse transcriptase was purchased from Promega Company, g) fluorescent quantitative PCR reaction solution an...

Embodiment 2

[0059] Example 2: Application of rabies virus fluorescent quantitative PCR kit in detection of rabies virus infection in cows

[0060] A). Kit composition:

[0061] a) The composition of the kit is as follows: (10 reactions)

[0062] RNA extraction solution A (5ml / tube) RNA extraction solution B (5ml / tube)

[0063] RNA extraction solution C (10ml / tube)

[0064] Filter column A and adsorption column B (sterile, RNase and DNase free)

[0065] Reverse transcription reaction solution (90μl / tube) Reverse transcriptase (10μl / tube)

[0066] RNase inhibitor (400U / tube)

[0067] Strong Positive Standard (100-fold diluted standard 20μl / tube, four tubes in total)

[0068] PCR reaction tube (sterile, RNase and DNase free) DEPC H 2 O (2ml / tube)

[0069] Negative standard (50μl / tube) Borderline positive standard (50μl / tube)

[0070] Fluorescent quantitative PCR reaction solution (125μl / tube)

[0071] (RNA extraction solution A, B, C and filter column adsorption column are products ...

Embodiment 3

[0090] Example 3: Application of rabies virus fluorescent quantitative PCR kit in detection of commercially available bovine serum

[0091] A). Kit composition:

[0092] a) The composition of the kit is as follows: (10 reactions)

[0093] RNA extraction solution A (4ml / tube) RNA extraction solution B (800μl / tube)

[0094] Reverse transcription reaction solution (90μl / tube) Reverse transcriptase (10μl / tube)

[0095] RNase inhibitor (400U / tube)

[0096] Strong Positive Standard (100-fold diluted standard 20μl / tube, four tubes in total)

[0097] PCR reaction tube (sterile, RNase and DNase free) DEPC H 2 O (2ml / tube)

[0098] Negative standard (50μl / tube) Borderline positive standard (50μl / tube)

[0099] Fluorescent quantitative PCR reaction solution (125μl / tube)

[0100] (RNA extraction solution A is a product of Invitrogen Company. b) reverse transcription reaction solution, d) reverse transcriptase was purchased from Promega Company, g) fluorescent quantitative PCR reactio...

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Abstract

The invention discloses a rabies virus detecting fluorescence quantitative PCR kit and an application thereof. The kit comprises: a) an RNA extraction solution, b) a reverse transcriptase reaction solution, c) reverse transcriptase, d) an RNA enzyme inhibitor, e) a primer and a TaqMan probe, f) a standard positive DNA template, and g) a PCR fluorescence quantitative reaction solution. The kit is characterized in that: primer sequence is a sense primer: 5'-TAGGATGCTATATGGGTCAAGTCAGA-3', and an antisense primer: 5'-TTCAAATGTCCCTTTCCCGAAGAA-3', and the size of an amplicon is 125 bp; the sequence of a fluorescence probe is: 5'-FAM-CAACGGTTATTGCTGCATGTGCTCCTGA-TAMRA-3', the 5' end of the probe is marked with a fluorescence emission group FAM, the 3' end is marked with a fluorescence quenching group TAMRA, the standard positive DNA template transforms a colon bacillus DH5 alpha by a pGEM-T carrier inserted with rabies virus N protein zone 391 bp segment, plasmid is extracted after proliferation, an A260 ration is measured in an ultraviolet spectrophotometer, and the plasmid is diluted by 10 times of gradient. The kit can efficiently and conveniently monitor the rabies virus pollution in serum products in real time, and can provide technical support for relevant fundamental researches, thus having quite broad application prospect.

Description

Technical field: [0001] The present invention relates to the field of biotechnology, more specifically to a fluorescent quantitative PCR kit for detecting rabies virus, and also relates to the use of the fluorescent quantitative PCR kit, which is suitable for real-time monitoring of rabies virus contamination in serum products, and at the same time for related Basic research provides technical support. Background technique [0002] Rabies virus belongs to the Rhabdoviridae family (Rhabdoviridae), lyssavirus (Lyssavirus), its nucleic acid is a single-stranded negative-sense RNA molecule without segments, rabies is an acute infectious disease caused by rabies virus, and can be used by both humans and animals Infection, also known as hydrophobia, mad dog disease, etc. The rabies virus is mainly transmitted among animals, and the disease is mainly infected by the rabies virus in the saliva carried on the teeth when the animal bites and invades the human or animal body. Once th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68G01N21/64C12R1/93
Inventor 郑从义郭佳徐国东张国荣
Owner WUHAN SANLI BIO TECH
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