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Fluorescence quantitative PCR kit for detecting calf diarrhea virus and application

A fluorescent quantitative and bovine diarrhea technology, applied in the direction of fluorescence/phosphorescence, microbial-based methods, microbial measurement/inspection, etc., can solve problems such as inability to calculate the starting DNA or RNA copy number

Active Publication Date: 2009-10-21
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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  • Fluorescence quantitative PCR kit for detecting calf diarrhea virus and application
  • Fluorescence quantitative PCR kit for detecting calf diarrhea virus and application

Examples

Experimental program
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Effect test

Embodiment 1

[0030] The fluorescent quantitative PCR kit composition and reaction condition thereof of embodiment 1 bovine diarrhea virus

[0031] A). Kit composition:

[0032] a) The composition of the kit is as follows: (10 reactions)

[0033] RNA extraction solution A (4ml / tube) RNA extraction solution B (800μl / tube)

[0034] Reverse transcription reaction solution (90μl / tube) Reverse transcriptase (10μl / tube)

[0035] RNase inhibitor (400U / tube)

[0036] Strong Positive Standard (100-fold diluted standard 20μl / tube, four tubes in total)

[0037] PCR reaction tube (sterile, RNase and DNase free) DEPC H 2 O (2ml / tube)

[0038] Negative standard (50μl / tube) Borderline positive standard (50μl / tube)

[0039] Fluorescent quantitative PCR reaction solution (125μl / tube)

[0040] (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 so...

Embodiment 2

[0060] Example 2: Application of Bovine Diarrhea Virus Fluorescence Quantitative PCR Kit in BVDV Epidemiological Investigation

[0061] A). Kit composition:

[0062] a) The composition of the kit is as follows: (10 reactions)

[0063] RNA extraction solution A (5ml / tube) RNA extraction solution B (5ml / tube)

[0064] RNA extraction solution C (10ml / tube)

[0065] Filter column A and adsorption column B (sterile, RNase and DNase free)

[0066] Reverse transcription reaction solution (90μl / tube) Reverse transcriptase (10μl / tube)

[0067] RNase inhibitor (400U / tube)

[0068] Strong Positive Standard (100-fold diluted standard 20μl / tube, four tubes in total)

[0069] PCR reaction tube (sterile, RNase and DNase free) DEPC H 2 O (2ml / tube)

[0070] Negative standard (50μl / tube) Borderline positive standard (50μl / tube)

[0071] Fluorescent quantitative PCR reaction solution (125μl / tube)

[0072] (RNA extraction solution A, B, C and filter column adsorption column are products...

Embodiment 3

[0091] Example 3: Application of Bovine Diarrhea Virus Fluorescent Quantitative PCR Kit in Detection of Commercially Available Bovine Serum

[0092] A). Kit composition:

[0093] a) The composition of the kit is as follows: (10 reactions)

[0094] RNA extraction solution A (4ml / tube) RNA extraction solution B (800μl / tube)

[0095] Reverse transcription reaction solution (90μl / tube) Reverse transcriptase (10μl / tube)

[0096] RNase inhibitor (400U / tube)

[0097] Strong Positive Standard (100-fold diluted standard 20μl / tube, four tubes in total)

[0098] PCR reaction tube (sterile, RNase and DNase free) DEPC H2 O (2ml / tube)

[0099] Negative standard (50μl / tube) Borderline positive standard (50μl / tube)

[0100] Fluorescent quantitative PCR reaction solution (125μl / tube)

[0101] (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 PC...

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Abstract

The invention discloses a fluorescence quantitative PCR kit for detecting calf diarrhea virus and application. The kit comprises a) RNA extract, b) reverse transcriptase reaction liquid, c) reverse transcriptase, d) RNA enzyme inhibitor, e) primers and TaqMan probe, f) standard positive DNA template, and g) fluorescence quantitative PCR reaction liquid. The kit is characterized in that in a positive primer, a negative primer and a fluorescence probe sequence, a 5' end of the probe marks a fluorescence emitting group FAM, a 3' end of the probe marks a fluorescence quenching group TAMRA, the standard positive DNA template converts colon bacillus DH5a by a pGEM-T carrier inserted into a 185bp fragment of a calf diarrhea virus 5'-UTR area, plasmids are extracted after multiplication, and A260 is measured by an ultraviolet spectrophotometer to definite quantity and is diluted by 10 times of gradient. Preparation for the kit comprises the following steps: A, treatment of a specimen and a standard product; and B, RT-PCR amplification by a two-step method and real-time fluorescence detection, and application of the fluorescence quantitative PCR kit in medicaments for quantitatively detecting calf diarrhea virus. The quantitative result is more accurate, reliable and stable, and the repeatability is good.

Description

Technical field: [0001] The invention relates to the field of biotechnology, more specifically to a fluorescent quantitative PCR kit for detecting bovine diarrhea virus, and also to the use of the fluorescent quantitative PCR kit, which can efficiently and conveniently detect bovine blood products and bovine serum Quantitative detection and quality monitoring can also be carried out for epidemiological investigation of bovine viral diarrhea-mucosal disease, and technical support can be provided for related basic research, and the application prospect is very broad. Background technique [0002] Bovine diarrhea virus belongs to Pestivirus (Pestivirus), Flaviviridae (Flaviviridae), is the smallest enveloped RNA virus, mainly causes bovine viral diarrhea-mucosal disease, and is a common disease in dairy and beef cattle herds. The disease mainly occurs in cattle, calves are more susceptible. The prevalence of antibodies in the herd is extremely high, so mild or insidious infect...

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 郑从义郭佳黄璇张国荣
Owner WUHAN SANLI BIO TECH
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