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Fluorescence quantitative RT-PCR (Reverse Transcription-Polymerase Chain Reaction) detection method for rabbit hemorrhagic disease virus

A rabbit hemorrhagic disease virus, fluorescent quantitative technology, applied in the field of rabbit hemorrhagic disease virus fluorescent quantitative RT-PCR detection, molecular detection of rabbit hemorrhagic disease virus, can solve the problem that the result judgment is easily affected by various factors, poor repeatability, processing Time-consuming and other problems, to achieve the effect of avoiding false positive results, easy to operate, and simple operation

Inactive Publication Date: 2011-08-17
CHINESE ACAD OF INSPECTION & QUARANTINE
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, there have been studies on the application of molecular biology to the diagnosis of RHDV, which mainly include the detection of RHDV by slide agglutination test (SHA), the detection of RHDV by enzyme-linked immunosorbent assay (ELISA), and the detection of RHDV by RT-PCR. The method has problems such as low sensitivity, poor repeatability, easily affected by various factors, and time-consuming processing.

Method used

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  • Fluorescence quantitative RT-PCR (Reverse Transcription-Polymerase Chain Reaction) detection method for rabbit hemorrhagic disease virus
  • Fluorescence quantitative RT-PCR (Reverse Transcription-Polymerase Chain Reaction) detection method for rabbit hemorrhagic disease virus
  • Fluorescence quantitative RT-PCR (Reverse Transcription-Polymerase Chain Reaction) detection method for rabbit hemorrhagic disease virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] The preparation of embodiment 1 positive standard

[0043] 1) RHDV virus extracts RNA, uses nucleotide sequence as the primer shown in SEQ ID No.4 and 5, carries out one-step RT-PCR amplification, obtains 240bp object fragment (such as figure 1 shown);

[0044] The PCR reaction system is:

[0045] Each 25 μL reaction solution contains: 10×RT-PCR buffer 2.5 μL, ultrapure dNTP mixture (10 mM each) 1 μL, 5×RT-PCR enhancer 5 μL, 40U / μL RNase inhibitor 0.25 μL, 2.5U / μL Hotmaster DNA polymerase 1.25 μL, Quant reverse transcriptase 0.25 μL, 10 mmol / L upstream and downstream primers 0.5 μL each, RNA template 1 μL, RNase-freeddH 2 O supplemented to 25 μL;

[0046] The PCR reaction conditions are: 50°C, 30min, 94°C, 2min, 94°C, 1min, 51.7°C, 1min, 65°C, 1min, 35 cycles of amplification, 65°C, 10min;

[0047] 2) The target fragment was then cloned into the PGEM-T Easy vector (purchased from Promega), and transformed into the host cell Escherichia coli DH5α (purchased from Beij...

Embodiment 2

[0054] Example 2 standard curve drawing

[0055] According to GenBank (GenBank registration number: FJ794180) RHDV VP60 capsid protein gene sequence design and screening PCR primers, and confirmed the specificity of each primer, the nucleotide sequences of the primers as SEQ ID No.1 and 2. Primers were synthesized by Shanghai Yingjun Biotechnology Co., Ltd. The length of the amplified fragment of the primer pair is 96bp.

[0056] Using the positive standard obtained in Example 1 as a template, using primers with nucleotide sequences as shown in SEQ ID No.1 and 2 and fluorescent probe VP60p, a one-step RT-PCR method is used for detection. Test results such as image 3 shown. The reaction system in the above RT-PCR is:

[0057] Each 25 μL reaction solution contains: 2×Quant One Step Probe qRT-PCR Master Mix (Quant one-step fluorescent quantitative RT-PCR kit (probe method)) 12.5 μL, 2.5U / μL Hotmaster DNA polymerase 1 μL, 4U / μL Quant reverse transcriptase 0.35 μL, 10 μmol / L ...

Embodiment 3

[0060] Embodiment 3 is to the detection of rabbit liver tissue sample

[0061] 1. Extraction of RNA from rabbit liver tissue samples

[0062] Take 0.2 mL of a 100-fold dilution of RHDV isolates (purchased from Jiangsu Academy of Agricultural Sciences), and inject intramuscularly into 3-month-old unimmunized rabbits. After 3 days, some rabbits died. The liver tissues of 10 dead rabbits were collected, and the rabbit liver tissue samples were extracted using the RNAprep pure animal tissue total RNA extraction kit (purchased from Tiangen Biochemical Technology Co., Ltd.).

[0063] (1) Add 300μL lysate solution RL to 200mg liver tissue, thoroughly grind it into a homogenate, add 590μL RNase-free ddH 2 O and 10 μL proteinase K, mix well and treat at 56°C for 10 min.

[0064] (2) Centrifuge the obtained liquid at 12000 rpm for 5 min, and take the supernatant.

[0065] (3) Slowly add 200 μL of absolute ethanol to the supernatant, mix well, put the obtained solution and the precipi...

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Abstract

The invention provides a primer and a TaqMan probe for fluorescence quantitative RT-PCR (Reverse Transcription-Polymerase Chain Reaction) detection of a rabbit hemorrhagic disease virus. The invention also provides a kit comprising the primer and the probe, and a method for detecting the rabbit hemorrhagic disease virus by using the primer and the probe with one-step RT-PCR. Due to the adoption of a one-step RT-PCR detection technology, reverse transcription and quantitative PCR amplification are continuously completed in one tube, amplification and detection are performed in a totally-closedtube, the probability of pollution caused by multiple operation is reduced, operation is easy, the volume loss in the sampling process is reduced, the dependability of a result is ensured, all cDNA (complementary Deoxyribonucleic Acid) samples obtained by reverse transcription are used for amplifying, and higher sensitivity is achieved.

Description

technical field [0001] The invention relates to a molecular detection method of rabbit hemorrhagic disease virus, in particular to a fluorescent quantitative RT-PCR detection method of rabbit hemorrhagic disease virus, which belongs to the field of biotechnology. Background technique [0002] Rabbit hemorrhagic disease (RHD) is a highly contagious infectious disease caused by rabbit hemorrhagic disease virus (RHDV). Characterized by respiratory hemorrhage, liver necrosis, parenchymal organ edema, congestion and hemorrhagic changes, it is a devastating infectious disease in rabbits. All the tested rabbit breeds have shown susceptibility. Under natural conditions, RHDV mainly infects adult rabbits, and pups under 2 months of age generally do not become ill when they are naturally infected. The disease causes huge economic losses to the rabbit industry, and also seriously threatens endangered wild rabbit breeds. As a result, RHDV has attracted great attention from scholars at...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12R1/93
Inventor 贾广乐苏国清林祥梅武昱孜孙卫东
Owner CHINESE ACAD OF INSPECTION & QUARANTINE
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