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Rabbit hemorrhagic disease virus RT-PCR detection method

A technology of RT-PCR and rabbit hemorrhagic disease virus, which is applied in the direction of microorganism-based methods, biochemical equipment and methods, and microorganism measurement/inspection. Poor repeatability and other issues, to avoid false positive results, simple operation, easy to operate

Inactive Publication Date: 2013-06-12
CHINESE ACAD OF INSPECTION & QUARANTINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] 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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  • Rabbit hemorrhagic disease virus RT-PCR detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] The preparation of embodiment 1 positive standard

[0044] 1) Extract RNA from RHDV virus, and use primers with nucleotide sequences as shown in SEQ ID No.4 and 5 to perform one-step RT-PCR amplification to obtain a 240bp target fragment (such as figure 1 shown);

[0045] The PCR reaction system is:

[0046] Each 25 μL reaction liquid 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-free ddH 2 O supplemented to 25 μL;

[0047] 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;

[0048] 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...

Embodiment 2

[0055] Example 2 standard curve drawing

[0056] The PCR primers were designed and screened according to GenBank (GenBank registration number: FJ794180) RHDV VP60 capsid protein gene sequence, and the specificity of each primer was confirmed. The nucleotide sequences of the primers are shown in 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.

[0057] 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:

[0058] 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 transcri...

Embodiment 3

[0061] Embodiment 3 is to the detection of rabbit liver tissue sample

[0062] 1. Extraction of RNA from rabbit liver tissue samples

[0063] 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. Three days later, 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.).

[0064] (1) Add 300 μL Lysis Buffer RL to 200 mg of liver tissue, thoroughly grind it into a homogenate, add 590 μL RNase-free ddH 2 O and 10 μL proteinase K, mix well and treat at 56°C for 10 min.

[0065] (2) Centrifuge the obtained liquid at 12000rpm for 5min, and take the supernatant.

[0066] (3) Slowly add 200 μL of absolute ethanol to the supernatant, mix well, put the obtained solution and the pr...

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Abstract

The invention provides a primer used for rabbit hemorrhagic disease virus RT-PCR detection, and also provides a kit containing the primer, and a method for the rabbit hemorrhagic disease virus RT-PCR detection through using the primer. The method has the advantages of accuracy, reliability, high sensitivity and high specificity, and has good clinic application values.

Description

[0001] This application is a divisional application of the patent application with the application date of March 15, 2011, the application number: 201110062529.2, and the invention title of "Rabbit Hemorrhagic Disease Virus Fluorescent Quantitative RT-PCR Detection Method". technical field [0002] 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 [0003] 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 pu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
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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